Publications by authors named "Willian Fernando Zambuzzi"

34 Publications

Propolis from southeastern Brazil produced by Apis mellifera affects innate immunity by modulating cell marker expression, cytokine production and intracellular pathways in human monocytes.

J Pharm Pharmacol 2021 Mar;73(2):135-144

Department of Chemical and Biological Sciences, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

Objectives: Propolis is a bee-made product used for centuries due to its diverse biological properties, including its immunomodulatory action. This work aimed at investigating whether propolis may affect monocyte functions challenged with retinoic acid (RA), B subunit of Escherichia coli heat-labile enterotoxin (EtxB), human melanoma-associated antigen-1 (MAGE-1) and lipopolysaccharide (LPS).

Methods: Monocytes from healthy donors were treated with the stimuli separately or in the presence of propolis. Cell viability was evaluated by MTT assay, cell marker expression was assessed by flow cytometry, cytokine production by ELISA, gene expression by RT-qPCR.

Key Findings: Propolis alone maintained TLR-2, TLR-4, HLA-DR, CD40 and CD80 expression in the monocytes; however, its combination with either MAGE-1 or LPS decreased CD40 expression triggered by the stimuli. Propolis maintained RA action on cell marker expression. Propolis inhibited TNF-α (with either EtxB or MAGE-1) and IL-6 (with either RA or MAGE-1), and increased IL-10 (with MAGE-1) production. Propolis downmodulated LC3 expression induced by LPS. It also induced a lower NF-kB expression than control cells and its combination with RA induced a higher expression than the stimulus alone.

Conclusions: Propolis potentially affected innate immunity by downmodulating the monocytes pro-inflammatory activity.
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http://dx.doi.org/10.1093/jpp/rgaa023DOI Listing
March 2021

Cystatin-like protein of sweet orange (CsinCPI-2) modulates pre-osteoblast differentiation via β-Catenin involvement.

J Mater Sci Mater Med 2021 Mar 22;32(4):33. Epub 2021 Mar 22.

Department of Restorative Dentistry, School of Dentistry at Araraquara, Sao Paulo State University - UNESP, Araraquara, São Paulo, Brazil.

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.
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http://dx.doi.org/10.1007/s10856-021-06504-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985097PMC
March 2021

PI3K/AKT signaling drives titanium-induced angiogenic stimulus.

J Mater Sci Mater Med 2021 Jan 27;32(1):18. Epub 2021 Jan 27.

Institute of Biosciences of Botucatu, Department of Chemical and Biological Sciences, UNESP - São Paulo State University, Botucatu, São Paulo, Brazil.

Although osseointegration and clinical success of titanium (Ti)-implanted materials depend on neovascularization in the reactional peri-implant tissue, very little has been achieved considering the Ti-molecules release on the behavior of endothelial cells. To address this issue, we challenged endothelial cells (HUVECs) with Ti-enriched medium obtained from two types of commercial titanium surfaces [presenting or not dual-acid etching (DAE)] up to 72 h to allow molecular machinery analysis. Our data show that the Ti-enriched medium provokes significant stimulus of angiogenesis-related machinery in endothelial cells by upexpressing VEGFR1, VEGFR2, VEGF, eNOS, and iNOS genes, while the PI3K/Akt signaling pathway was also significantly enhanced. As PI3K/AKT signaling was related to angiogenesis in response to vascular endothelial growth factor (VEGF), we addressed the importance of PI3K/Akt upon Ti-enriched medium responses by concomitantly treating the cells with wortmannin, a well-known PI3K inhibitor. Wortmannin suppressed the angiogenic factors, because VEGF, VEGFR1, and eNOS genes were downregulated in those cells, highlighting the importance of PI3K/AKT signaling on driving angiogenic phenotype and angiogenesis performance within the peri-implant tissue reaction. In conjunction, these data reinforce that titanium-implantable devices modify the metabolism of surrounding cells, such as endothelial cells, probably coupling osteogenesis and angiogenesis processes in peri-implant tissue and then contributing to successfully osseointegration of biomedical titanium-based devices.
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http://dx.doi.org/10.1007/s10856-020-06473-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840643PMC
January 2021

Platelet microparticles load a repertory of miRNAs programmed to drive osteogenic phenotype.

J Biomed Mater Res A 2021 Aug 10;109(8):1502-1511. Epub 2020 Dec 10.

Department of Chemistry and Biochemistry, São Paulo State University (UNESP), Institute of Biosciences, campus Botucatu, São Paulo, Brazil.

Autologous platelet-rich plasma accelerates bone healing by releasing biomolecules during their degranulation process, which are transported by vesicle-like structures called platelet microparticles (PMPs). However, the underlying mechanisms regulating the osteogenic differentiation by PMP-released miRs remain poorly understood and this prompted us to better address this issue. Thus, miRNAseq expression profiles (E-GEOD-76789) were downloaded from ArrayExpress database. GEO2R was performed to evaluate the differential expression, and mirnatap R package was used to find targets for differentially expressed miRNAs. An extend protein-protein (ePPI) network for osteogenic marker proteins was generated using String, and DAVID tools were used to perform gene ontology and KEGG pathway analysis from ePPI and miRNAs targets. Our data show that ePPI network was composed by 232 nodes and 2,175 edges, with a clustering coefficient of 0.546. MCODE was able to identify seven clusters contained in the ePPI network, and the two that presented a score above 10 were used in further analysis. Conversely, 15,944 different targets were found as down-expressed while 5,715 different targets were up-expressed. Among the downregulated 75 miRNAs, 70 have predicted targets present in the ePPI network, while the 21 upregulated miRNAs have 19 predicted targets in the ePPI network. Our study provides a registry of miRNAs that play a central role in regulating osteogenic phenotype, which might have potential therapeutic applications in bone regeneration and bone tissue engineering.
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http://dx.doi.org/10.1002/jbm.a.37140DOI Listing
August 2021

Publisher Correction: Bisphosphonate-based surface biofunctionalization improves titanium biocompatibility.

J Mater Sci Mater Med 2020 Nov 27;31(11):112. Epub 2020 Nov 27.

Department of Physics, UNESP-São Paulo State University, School of Sciences, Bauru, Brazil.

A Correction to this paper has been published: https://doi.org/10.1007/s10856-020-06470-x.
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http://dx.doi.org/10.1007/s10856-020-06470-xDOI Listing
November 2020

Bisphosphonate-based surface biofunctionalization improves titanium biocompatibility.

J Mater Sci Mater Med 2020 Nov 7;31(11):109. Epub 2020 Nov 7.

Department of Physics, UNESP-São Paulo State University, School of Sciences, Bauru, Brazil.

Novel-biofunctionalized surfaces are required to improve the performance of endosseous implants, which are mainly related to the resistance against biocorrosion, as well as for the consideration of osteoinductive phenomena. Among different strategies, the use of bisphosphonate molecules as linkers between titanium dioxide (TiO) surfaces and proteins is a distinctive approach, one in which bisphosphonate could play a role in the osseointegration. Thus, to address this issue, we proposed a novel biofunctionalization of TiO surfaces using sodium alendronate (ALN) as a linker and bovine serum albumin as the protein. Physicochemical analysis of the functionalized surfaces was performed using contact angle analyses and surface roughness measurements, which indicated an efficient functionalization. The biocompatibility of the functionalized surfaces was analyzed through the adhesion behavior of the pre-osteoblasts onto the samples. Overall, our data showed a significant improvement concerning the cell adhesion by modulating the adhesion cell-related set of genes. The obtained results show that for modified surfaces there is an increase of up to 100 times in the percentage of cells adhered when compared to the control, besides the extracellular matrix remodeling seemed to be an essential prerequisite for the early stages of cell adhesion on to the biomaterials, which was assayed by evaluating the matrix metalloproteinase activities as well as the gene activations. In the expressions of the Bsp and Bglap2 genes, for the group containing ALN (TiO + ALN), it was observed an increase in expression (approximately sixfold change) when compared to the control. Altogether, our data clearly showed that the bisphosphonate-biofunctionalized surface enhanced the biocompatibility of titanium and claims to further progress preclinical in vivo experimentation.
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http://dx.doi.org/10.1007/s10856-020-06437-yDOI Listing
November 2020

Infraphysiological 17β-estradiol (E2) concentration compromises osteoblast differentiation through Src stimulation of cell proliferation and ECM remodeling stimulus.

Mol Cell Endocrinol 2020 12 8;518:111027. Epub 2020 Sep 8.

Experimental Research Unit, School of Medicine of Botucatu (FMB), Sao Paulo State University (UNESP), Botucatu, 18618-970, Sao Paulo State, Brazil.

It has been shown that 17β-estradiol (E2) helps to prevent bone loss. This study was undertaken to verify whether E2 action in human osteoblasts involves changes in the transcriptional profile of the TNF-α, IFN-γ, NF-κB, TRAIL, TGF-β, MMP2, MMP9, RECK, TIMP1, TIMP2, CDK2, CDK4, SRC, RUNX2, and SHH genes. Infraphysiological doses of E2 elevated mRNAs in all genes except for INF-γ, TRAIL, and TGF-β. Importantly, a significant increase in the CDKs -2 and -4 genes was found, which strongly suggests cell cycle progression, with a potential dependency of Src involvement, as well as a suppression of the osteoblast differentiation machinery, with ECM remodeling being involved. These data suggest that E2 plays an important role in bone formation and remodeling, and Src seems to play a pivotal role in driving cell proliferation and ECM remodeling. Taken together, these findings contribute to an understanding of the effects of infraphysiological E2 on modulating bone homeostasis, favoring bone resorption, and leading to osteoporosis.
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http://dx.doi.org/10.1016/j.mce.2020.111027DOI Listing
December 2020

Biofunctionalization of titanium surfaces with alendronate and albumin modulates osteoblast performance.

Heliyon 2020 Jul 21;6(7):e04455. Epub 2020 Jul 21.

Advanced Materials and Nanotechnology Laboratory - UNESP - São Paulo State University School of Sciences, Department of Physics, Bauru, Brazil.

Background: Biofunctionalization of titanium surfaces can improve host responses, especially considering the time for osteointegration and patient recovery. This prompted us to modify titanium surfaces with alendronate and albumin and to investigate the behavior of osteoblasts on these surfaces.

Methods: The biofunctionalization of titanium surfaces was characterized using classical physicochemical approaches and later used to challenge pre-osteoblast cells up to 24 h. Then their viability and molecular behavior were investigated using mitochondrial dehydrogenase activity and RTq-PCR technologies, respectively. Potential stimulus of extracellular remodeling was also investigated by zymography.

Results: Our data indicates a differential behavior of cells responding to the surfaces, considering the activity of mitochondrial dehydrogenases. Molecularly, the differential expression of genes related with cell adhesion highlighted the importance of , and . These 3 genes were significantly decreased in response to titanium surfaces modified with alendronate, but this behavior was reverted when alendronate was associated with albumin. Alendronate-modified surfaces promoted a significant increase on ECM remodeling, as well as culminating with greater gene activity related to the osteogenic phenotype (, , ).

Conclusion: Altogether, our study found interesting osteogenic behavior of cells in response to alendronate and albumin surfaces, which indicates the need for analyses to better consider these surfaces before clinical trials within the biomedical field.
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http://dx.doi.org/10.1016/j.heliyon.2020.e04455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378701PMC
July 2020

Obesity/Metabolic Syndrome and Diabetes Mellitus on Peri-implantitis.

Trends Endocrinol Metab 2020 08 23;31(8):596-610. Epub 2020 Jun 23.

Department of Biomaterials and Biomimetics, New York University College of Dentistry, New York, NY, USA; Hansjörg Wyss Department of Plastic Surgery, NYU Langone Health School of Medicine, New York, NY, USA; Department of Mechanical and Aerospace Engineering, NYU Tandon School of Engineering, Brooklyn, NY, USA. Electronic address:

Literature has reported that up to 50% of dental implants may be affected by peri-implantitis, a bacteria-induced chronic inflammatory process, which promotes osteoclast-mediated bone resorption and inhibits bone formation, leading to progressive bone loss around implants. Current evidence points toward an increased risk for the development of peri-implantitis in both obesity/metabolic syndrome (MetS) and diabetes mellitus (DM) conditions relative to the healthy population. Currently, there is no effective treatment for peri-implantitis and the 50% prevalence in MetS and DM, along with its predicted increase in the worldwide population, presents a major concern in implant dentistry as hyperglycemic conditions are associated with bone-healing impairment; this may be through dysfunction of osteocalcin-induced glucose metabolism. The MetS/DM proinflammatory systemic condition and altered immune/microbiome response affect both catabolic and anabolic events of bone-healing that include increased osteoclastogenesis and compromised osteoblast activity, which could be explained by the dysfunction of insulin receptor that led to activation of signals related with osteoblast differentiation. Furthermore, chronic hyperglycemia along with associated micro- and macro-vascular ailments leads to delayed/impaired wound healing due to activation of pathways that are particularly important in initiating events linked to inflammation, oxidative stress, and cell apoptosis; this may be through deactivation of AKT/PKB protein, which possesses a pivotal role in drive survival and eNOS signaling. This review presents an overview of the local and systemic mechanisms synergistically affecting bone-healing impairment in MetS/DM individuals, as well as a rationale for hierarchical animal model selection, in an effort to characterize peri-implantitis disease and treatment.
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http://dx.doi.org/10.1016/j.tem.2020.05.005DOI Listing
August 2020

Fibroblast-secreted trophic factors contribute with ECM remodeling stimulus and upmodulate osteocyte gene markers in osteoblasts.

Biochimie 2020 Jan 30;168:92-99. Epub 2019 Oct 30.

Lab. of Bioassays and Cell Dynamics, Department of Chemistry and Biochemistry, Institute of Biosciences, Universidade Estadual Paulista, UNESP, CEP 18618-970, Botucatu, São Paulo, Brazil. Electronic address:

As osteogenesis is a multifactorial mechanism, we wonder whether osteoblast-induced extracellular matrix (ECM) remodeling might be modulated by trophic factors released by fibroblasts in a paracrine signaling manner. To address this issue, fibroblasts were cultured for 72 h under conventional conditions when their conditioned medium was harvested and used to challenge pre-osteoblasts (MC3T3-E1 cells) for 14 days. Preliminarily, we validated the potential effect of fibroblasts in contributing to osteocyte phenotype, which specifically requires significant expression of Dentin Matrix Protein 1 (DMP1; about 10-fold changes) and Sclerostin (SOST; about 7-fold changes), both biomarkers of osteocyte. Fibroblasts also seem contributing to ECM remodeling in osteoblasts, because we detected a high level of both mRNA and enzyme activities of matrix metalloproteinase -9 (MMP-9) as well as a high level of reversion inducing cysteine rich protein with kazal motifs (RECK) transcripts (about 13-fold changes), a membrane-anchored MMP inhibitor, which seems to be a constitutive pathway in osteoblasts. Considering inflammatory panorama and using RTqPCR technology, both IL-13 (about 13-fold changes) and IL-33 (about 5-fold changes) genes were up-expressed in response to the fibroblast-secreted trophic factors, as were the receptor activator of NF-κB ligand (RANKL; about 8-fold changes) and osteoprotegerin (OPG; about 3-fold changes). Although preliminary, these data suggest a stimulus to finely control osteoclastogenesis, and this mechanism reinforces the role of fibroblasts in bone remodeling and homeostasis. Moreover, these results suggest an important crosstalk between fibroblast and osteoblast, when fibroblast-secreted trophic factors upmodulate osteocyte gene markers and contribute to ECM remodeling stimulus in osteoblast.
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http://dx.doi.org/10.1016/j.biochi.2019.10.013DOI Listing
January 2020

Updating the role of matrix metalloproteinases in mineralized tissue and related diseases.

J Appl Oral Sci 2019 Sep 9;27:e20180596. Epub 2019 Sep 9.

Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Ciências Biológicas, Laboratório de Bioquímica, Bauru, São Paulo, Brasil.

Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of "smart" medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer.
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http://dx.doi.org/10.1590/1678-7757-2018-0596DOI Listing
September 2019

In vivo and in vitro anti-inflammatory and pro-osteogenic effects of citrus cystatin CsinCPI-2.

Cytokine 2019 11 18;123:154760. Epub 2019 Jun 18.

Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Sao Paulo State University - UNESP, Araraquara, São Paulo, Brazil. Electronic address:

Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (p < 0.05) on cathepsin K, cathepsin B, IL-1β, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (p < 0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.
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http://dx.doi.org/10.1016/j.cyto.2019.154760DOI Listing
November 2019

Cobalt-chromium-enriched medium ameliorates shear-stressed endothelial cell performance.

J Trace Elem Med Biol 2019 Jul 24;54:163-171. Epub 2019 Apr 24.

Department of Chemistry and Biochemistry, Bioscience Institute, Sao Paulo State University, UNESP, Campus Botucatu, Botucatu, São Paulo, Brazil; Electron Microscopy Center, IBB, UNESP, Botucatu, SP, Brazil. Electronic address:

Angiogenesis is a relevant mechanism to be considered for the success of bone healing, even considering endosseous implantable devices, providing adequate delivery of substances necessaries for the cell viability and bone de novo deposition. Within of the repertory of metal-based implantable alloys, cobalt-chromium (CoCr) has emerged with very interesting properties for biomedical applications. Additionally, we have shown that released molecules from implants devices are able to modulate cells away and because that we hypothesized these released molecules might act on endothelial cells. In order to better address this issue, we investigated the effect of Co-Cr-enriched medium on endothelial cells (HUVECs), considering a biological model subjecting those cells to shear-stress to partially mimic the physiological environment and further allow investigating intracellular pathways responsible to drive cytoskeletal rearrangement, cell viability and extracellular matrix (ECM) remodeling processes. Considering the analysis of the metalloproteinases (MMPs) activities, our data indicates an intense ECM remodeling in response to CoCr-enriched medium suggesting some role on angiogenesis once ECM remodeling is prerequisite to cell growth. This was better addressed by revealing its involvement on modifying both mRNA expression and protein levels of members of the MAPK family. Additionally, the expression of CDK4 gene was modulated within the cell response to Co-Cr-enriched medium, while the modulation in the expression of P15 and P21 indicates an important regulatory mechanism required. Overall, our results demonstrate that trace of CoCr elements triggers decisive intracellular signaling in shear-stressed endothelial cells, suggesting influence on angiogenesis-related mechanism and they bring novel insights to explain the biological activity of CoCr as it has been emerged as interesting biomedical materials within the medical and dentistry fields.
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http://dx.doi.org/10.1016/j.jtemb.2019.04.012DOI Listing
July 2019

A new chemotherapeutic approach using doxorubicin simultaneously with geopropolis favoring monocyte functions.

Life Sci 2019 Jan 29;217:81-90. Epub 2018 Nov 29.

São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Brazil. Electronic address:

Aims: Chemotherapy has been widely used to treat cancer although it may affect non-target cells involved in the immune response. This work aimed at elucidating whether the chemotherapeutic agent doxorubicin in combination with geopropolis produced by Melipona fasciculata Smith could affect nontumor immune cells, evaluating their immunomodulatory effects on human monocytes.

Main Methods: Cell viability, expression of cell markers (HLA-DR, TLR-2, TLR-4, C80 and CD40), cytokine production (TNF-α, IL-1β, IL-6 and IL-10), intracellular pathways (NF-κB and autophagy), the microbicidal activity of monocytes and hydrogen peroxide (HO) production were analyzed.

Key Findings: Data showed that doxorubicin + geopropolis diminished IL-6 secretion, stimulated TNF-α and IL-10 production, TLR-4 and CD80 expression, NF-κB and autophagy pathway, as well as the bactericidal activity.

Significance: Our findings revealed a new chemotherapeutic approach using doxorubicin simultaneously with geopropolis without affecting human monocytes viability and exerting immunomodulatory effects, favoring cell functions. While doxorubicin altered some immunological parameters, the addition of geopropolis compensated some changes.
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http://dx.doi.org/10.1016/j.lfs.2018.11.060DOI Listing
January 2019

Titanium-enriched medium drives low profile of ECM remodeling as a pre-requisite to pre-osteoblast viability and proliferative phenotype.

J Trace Elem Med Biol 2018 Dec 20;50:339-346. Epub 2018 Jul 20.

Dept. of Chemistry and Biochemistry, Bioscience Institute, São Paulo State University, UNESP, Campus Botucatu, Botucatu, São Paulo, Brazil; Electron Microscopy Center, IBB, UNESP, Botucatu, SP, Brazil. Electronic address:

Titanium is widely used for biomedical applications, but little information is being delivered regarding the cellular/molecular mechanisms explaining their efficacy, mainly considering the effects of the Ti-released trace elements on pre-osteoblasts. We addressed this issue by investigating decisive intracellular signal transduction able to modulate cytoskeleton rearrangement, proliferative phenotype and extracellular matrix (ECM) remodeling. We considered titanium grades IV and V, submitted or not to dual acid-etching (w/DAE or wo/DAE, respectively). Our results showed there is no cytotoxicity, preserving AKT involvement. Additionally, Ti-enriched medium promoted a diminution of the downstream signaling upon integrin activation (phosphorylating Rac1 and cofilin), guaranteeing a dynamic cytoskeleton rearrangement. Moreover, the low profile of ECM remodeling obtained in response to trace molecules released by Ti-based devices seems contributing to the osteoblast performance in mediating extracellular support to cell anchorage. This hypothesis was validated by the up-expression of ß1-integrin, src and Focal adhesion kinase (fak) genes, mainly in response to titanium grade V. Proliferative phenotype showed an unbalance between cyclin-dependent kinases (CDKs) and p15/p21. In conjunction, we showed for the first time that trace elements from Ti-based biomedical devices provoke important modulation of the osteoblast biology, driving cell anchoring, viability, and proliferative phenotype. Certainly, these biological outcomes compromise implant osseointegration.
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http://dx.doi.org/10.1016/j.jtemb.2018.07.015DOI Listing
December 2018

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production, characterization and application.

Prep Biochem Biotechnol 2018 14;48(7):565-573. Epub 2018 Jun 14.

a Chemistry and Biochemistry Department, Institute of Biosciences , São Paulo State University (UNESP) , Botucatu , SP , Brazil.

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58 U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91 U/g of total lipases produced on average by the 15 microorganisms. 01/frit (33.53 U/g) and (INCQS 40015)/frit (34.76 U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl--pierylhydrazyl) radical reduction and antibacterial activity against , , , Enteritidis and , as well as cytotoxicity to SCC9 cells (squamous cancer cells).
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http://dx.doi.org/10.1080/10826068.2018.1476879DOI Listing
June 2018

Fluoride affects bone repair differently in mice models with distinct bone densities.

J Trace Elem Med Biol 2017 Jan 13;39:129-134. Epub 2016 Sep 13.

Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Al. Octávio Pinheiro Brisolla, 9-75, 17012-901, Bauru, São Paulo, Brazil. Electronic address:

We grouped mice [strains: C57BL/6J (n=32) and C3H/HeJ (n=32)] to address the influence of bone density on fluoride's (F's) biological effects. These animals received low-fluoride food and water containing 0 (control group) or 50ppm of F for up to 28days. The upper left central incisor was extracted, and the left maxilla was collected at 7, 14, 21, and 28days for histological and histomorphometric analysis to estimate bone neoformation. Our results showed bone neoformation in all of the evaluated groups, with the presence of bone islets invading the center of the alveoli when replacing the existing connective tissue. Curiously, this biological phenomenon was more evident in the C57BL/6J strain. The histomorphometric analysis confirmed the histological findings in relation to the amount of new bone tissue and showed a decrease in C3H/HeJ mice (control group). Altogether, our results showed differential effects of fluoride bone metabolism, confirming a genetic component in susceptibility to the effects of fluoride.
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http://dx.doi.org/10.1016/j.jtemb.2016.09.004DOI Listing
January 2017

Genetic Variants in Folate and Cobalamin Metabolism-Related Genes in Nonsyndromic Cleft Lip and/or Palate.

Braz Dent J 2015 Nov-Dec;26(6):561-5

Department of Pediatric Clinic, School of Dentistry of Ribeirão Preto, USP - University of São Paulo, Ribeirão Preto, SP, Brazil.

The aim of this study was to evaluate the association of the polymorphisms in TCN2 (rs1801198) gene and in MTRR (rs1801394) gene with nonsyndromic cleft lip and/or palate (NSCL/P) in a Brazilian population. Genomic DNA was extracted from buccal cells. The polymorphisms in TCN2 (rs1801198) and MTRR (rs1801394) genes were genotyped by carrying out real-time PCR and Taqman assay. Chi-square test was used to determine the association between genotype and allele frequencies with NSCL/P and NSCL/P subgroups (cleft lip only, cleft lip and palate, and cleft palate only). Eight hundred and sixty seven unrelated individuals (401 cases with NSCL/P and 466 individuals without cleft) were evaluated. Genotype distributions of TCN2 and MTRR polymorphisms were in Hardy-Weinberg equilibrium. The TCN2 polymorphic genotype GG was identified in 16.7% of the NSCL/P group and in 14.1% of the non-cleft group (p>0.05). Similarly, the frequency of MTRR genotype (GG) was similar in NSCL/P group (15.5%) and control group (17.8%) (p>0.05). Multivariate analysis showed an association between MTRR and the subgroup that the mother smoked during pregnancy (p=0.039). Our findings did not demonstrate an association between TCN2 polymorphisms and NSCL/P, however suggests an association between MTRR and NSCL/P etiology.
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http://dx.doi.org/10.1590/0103-6440201300394DOI Listing
February 2017

Bone Morphogenetic Proteins: Promising Molecules for Bone Healing, Bioengineering, and Regenerative Medicine.

Vitam Horm 2015 15;99:293-322. Epub 2015 Jul 15.

Bioengineering Division, National Institute of Metrology, Quality, and Technology, Duque de Caxias, Brazil; Department of Dental Materials, Dental School, Fluminense Federal University, Niteroi, Brazil. Electronic address:

Bone morphogenetic proteins (BMPs), glycoproteins secreted by some cells, are members of the TGF-β superfamily that have been implicated in a wide variety of roles. Currently, about 20 different BMPs have been identified and grouped into subfamilies, according to similarities with respect to their amino acid sequences. It has been shown that BMPs are secreted growth factors involved in mesenchymal stem cell differentiation, also being reported to control the differentiation of cancer stem cells. BMPs initiate signaling from the cell surface by binding to two different receptors (R: Type I and II). The heterodimeric formation of type I R and II R may occur before or after BMP binding, inducing signal transduction pathways through SMADs. BMPs may also signal through SMAD-independent pathways via mitogen-activated protein kinases (ERK, p38MAPKs, JNK). BMPs may act in an autocrine or paracrine manner, being regulated by specific antagonists, namely: noggin and chordin. Genetic engineering allows the production of large amounts of BMPs for clinical use, and clinical trials have shown the benefits of FDA-approved recombinant human BMPs 2 and 7. Several materials from synthetic to natural sources have been tested as BMP carriers, ranging from hydroxyapatite, and organic polymers to collagen. Bioactive membranes doped with BMPs are promising options, acting to accelerate and enhance osteointegration. The development of smart materials, mainly based on biopolymers and bone-like calcium phosphates, appears to provide an attractive alternative for delivering BMPs in an adequately controlled fashion. BMPs have revealed a promising future for the fields of Bioengineering and Regenerative Medicine. In this chapter, we review and discuss the data on BMP structure, mechanisms of action, and possible clinical applications.
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http://dx.doi.org/10.1016/bs.vh.2015.06.002DOI Listing
May 2016

Cellular behavior as a dynamic field for exploring bone bioengineering: a closer look at cell-biomaterial interface.

Arch Biochem Biophys 2014 Nov 27;561:88-98. Epub 2014 Jun 27.

Laboratório de Bioensaios e Dinâmica Celular, Depto. Química e Bioquímica, Instituto de Biociência, Universidade Estadual Paulista, UNESP, Campus Botucatu, Botucatu, SP, Brazil. Electronic address:

Bone is a highly dynamic and specialized tissue, capable of regenerating itself spontaneously when afflicted by minor injuries. Nevertheless, when major lesions occur, it becomes necessary to use biomaterials, which are not only able to endure the cellular proliferation and migration, but also to substitute the original tissue or integrate itself to it. With the life expectancy growth, regenerative medicine has been gaining constant attention in the reconstructive field of dentistry and orthopedy. Focusing on broadening the therapeutic possibilities for the regeneration of injured organs, the development of biomaterials allied with the applicability of gene therapy and bone bioengineering has been receiving vast attention over the recent years. The progress of cellular and molecular biology techniques gave way to new-guided therapy possibilities. Supported by multidisciplinary activities, tissue engineering combines the interaction of physicists, chemists, biologists, engineers, biotechnologist, dentists and physicians with common goals: the search for materials that could promote and lead cell activity. A well-oriented combining of scaffolds, promoting factors, cells, together with gene therapy advances may open new avenues to bone healing in the near future. In this review, our target was to write a report bringing overall concepts on tissue bioengineering, with a special attention to decisive biological parameters for the development of biomaterials, as well as to discuss known intracellular signal transduction as a new manner to be explored within this field, aiming to predict in vitro the quality of the host cell/material and thus contributing with the development of regenerative medicine.
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http://dx.doi.org/10.1016/j.abb.2014.06.019DOI Listing
November 2014

Periosteal-derived cells for bone bioengineering: a promising candidate.

Clin Oral Implants Res 2012 Oct 4;23(10):1238-42. Epub 2012 Jan 4.

Cell Therapy Center, University Hospital Antonio Pedro, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil.

Purpose: Over the last years so many efforts have been made in order to indentify natural sources of osteogenic cells for the success of bone bioengineering. Among them, periosteum tissue has emerged as an interesting candidate. Thus, we decided to evaluate the osteogenic potential of periosteal-derived cells by describing a sequence of biological events since initial morphological changes to mineralization of extracellular matrix (ECM).

Methods: Periosteal-derived cells were obtained from calvarial of adult rats. After the primary culture and expansion, the adherent cells were cultured at 7, 14, 21 and 28 days under a classical osteogenic culture medium in order to evaluate the differentiation of those cells in mature osteoblast. It was monitored by evaluating a time-line of alkaline phosphatase (ALP) activity (biomarker of osteoblast differentiation) and afterwards nodules of mineralization (measured by von Kossa staining and calcium content).

Results: Analysis from phase-contrast microscopy revealed mainly morphological changes ranging since fibroblast-shaped (7 days, semi-confluent culture at exponential growth) to polyhedral-shaped cells (14-28 days, confluent culture during differentiation process). ALP activity was linearly increased since 14-28 days while amount of protein remained unchanged. Interesting, our data from von Kossa staining reveled a highest incidence of mineralization nodules at 28 days.

Conclusion: Taken our results together, we can suggest that periosteal-derived cells present an interesting potential to differentiate in mature osteoblast able to promote mineralization in vitro by incorporating to ECM circulating calcium from extracellular compartment. From our point of view, this source of osteogenic cells can be explored by bioengineers in order to advance therapeutic protocols able to solve bone degenerative lesions.
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http://dx.doi.org/10.1111/j.1600-0501.2011.02287.xDOI Listing
October 2012

Biological monitoring of a promissory xenogenic pin for biomedical applications: a preliminary intraosseous study in rats.

Clin Oral Implants Res 2012 Mar 23;23(3):367-72. Epub 2011 Mar 23.

Cell Therapy Center (UPC), University Hospital Antonio Pedro, Fluminense Federal University, Niterói, Rio de Janeiro.

Objectives: Over the last years, it is known that in some cases metal devices for biomedical applications present some disadvantages suggesting absorbable materials (natural or synthetic) as an alternative of choice. Here, our goal was to evaluate the biological response of a xenogenic pin, derived from bovine cortical bone, intraosseously implanted in the femur of rats.

Material And Methods: After 10, 14, 30 and 60 days from implantation, the animals (n=5/period) were killed and the femurs carefully collected and dissected out under histological demands. For identifying the osteoclastogenesis level at 60 days, we performed the immunohistochemisty approach using antibody against RANKL.

Results: Interestingly, our results showed that the incidence of neutrophils and leukocytes was observed only at the beginning (10 days). Clear evidences of pin degradation by host cells started at 14 days and it was more intensive at 60 days, when we detected the majority of the presence of giant multinucleated cells, which were very similar to osteoclast cells contacting the implanted pin. To check osteoclastogenesis at 60 days, we evaluated RANKL expression and it was positive for those resident multinucleated cells while a new bone deposition was verified surrounding the pins in all evaluated periods.

Conclusions: Altogether, our results showed that pins from fully processed bovine bone are biocompatible and absorbable, allowing bone neoformation and it is a promissory device for biomedical applications.
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http://dx.doi.org/10.1111/j.1600-0501.2010.02143.xDOI Listing
March 2012

Biological monitoring of a xenomaterial for grafting: an evaluation in critical-size calvarial defects.

J Mater Sci Mater Med 2011 Apr 20;22(4):997-1004. Epub 2011 Mar 20.

Department of Endodontics, University of Grande Rio, Duque de Caxias, RJ 25071-202, Brazil.

Our purpose was to evaluate the osteoconduction potential of mixed bovine bone (MBB) xenografts as an alternative for bone grafting of critical-size defects in the calvaria of rats. After surgery, in the time intervals of 1, 3, 6, and 9 months, rats were killed and their skulls collected, radiographed and histologically prepared for analysis. The data obtained from histological analysis reported that the particles of MBB did not promote an intense immunological response, evidencing its biocompatibility in rats. Our results clearly showed the interesting evidence that MBB was not completely reabsorbed at 9 months while a small amount of newly formed bone was deposited by osteoprogenitor cells bordering the defect. However, this discrete bone-forming stimulation was unable to regenerate the bone defect. Overall, our results suggest that the properties of MBB are not suitable for stimulating intense bone regeneration in critical bone defects in rats.
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http://dx.doi.org/10.1007/s10856-011-4278-7DOI Listing
April 2011

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

Cell Tissue Res 2010 Apr 19;340(1):61-9. Epub 2010 Feb 19.

Department of Biochemistry, IB, University of Campinas, Campinas, SP, Brazil.

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.
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http://dx.doi.org/10.1007/s00441-010-0931-6DOI Listing
April 2010

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

J Mol Histol 2009 Jun 17;40(3):201-7. Epub 2009 Oct 17.

Laboratory of Molecular Pathology, Department of Oral Pathology, Dental School, University of São Paulo, Sao Paulo, SP, Brazil.

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.
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http://dx.doi.org/10.1007/s10735-009-9231-4DOI Listing
June 2009

On the road to understanding of the osteoblast adhesion: cytoskeleton organization is rearranged by distinct signaling pathways.

J Cell Biochem 2009 Sep;108(1):134-44

Department of Biochemistry, Biology Institute, University of Campinas, Campinas, São Paulo, Brazil.

Pre-osteoblast adhesion attracts increasing interest in both medicine and dentistry. However, how this physiological event alters osteoblast phenotype is poorly understood. We therefore attempted to address this question by investigating key biochemical mechanism that governs pre-osteoblast adhesion on polystyrene surface. Importantly, we found that cofilin activity was strongly modulated by PP2A (Ser/Thr phosphatase), while cell-cycle was arrested. Accordingly, we observed that the profile of cofilin phosphorylation (at Ser03) was similar to phospho-PP2A (at Tyr307). Also, it is plausible to suggest during pre-osteoblast adhesion that PP2A phosphorylation at Y307 was executed by phospho-Src (Y416). In addition, it was observed that MAPKp38, but not MAPK-erk, played a key role on pre-osteoblast adhesion by phosphorylating MAPKAPK-2 and ATF-2 (also called CRE-BP1). Also, the up-modulation of RhoA reported here suggests its involvement at the beginning of osteoblast attachment, while Akt remained active during all periods. Altogether, our results clearly showed that osteoblast adhesion is under an intricate network of signaling molecules, which are responsible to guide their interaction with substrate mainly via cytoskeleton rearrangement.
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http://dx.doi.org/10.1002/jcb.22236DOI Listing
September 2009

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation.

Arch Oral Biol 2009 Jul 2;54(7):642-50. Epub 2009 May 2.

Department of Biological Sciences, Bauru Dental School, University of São Paulo, Bauru, SP, Brazil.

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels.

Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays.

Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter.

Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. In this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation.
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http://dx.doi.org/10.1016/j.archoralbio.2009.03.011DOI Listing
July 2009

Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells.

Biochimie 2008 Jun 3;90(6):843-54. Epub 2008 Feb 3.

Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Cidade Universitária, Zeferino Vaz, Barão Geraldo, 13083-970 Campinas, SP, Brazil.

Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular basis for these actions remains only partly understood and hence we investigated the effects of ferruginol on androgen-independent human prostate cancer cells (PC3), a known model for solid tumor cells with an exceptional resistance to therapy. The results show that ferruginol induces PC3 cell death via activation of caspases as well as apoptosis-inducing factor (AIF) as confirmed by its translocation into the nucleus. In order to clarify the biochemical mechanism responsible for the anti-tumor activity of ferruginol, we analyzed a set of molecular mediators involved in tumor cell survival, progression and aggressiveness. Ferruginol was able to trigger inhibition/downregulation of Ras/PI3K, STAT 3/5, protein tyrosine phosphatase and protein kinases related to cell cycle regulation. Importantly, the toxic effect of ferruginol was dramatically impeded in a more reducing environment, which indicates that at least in part, the anti-tumoral activity of ferruginol might be related to redox status modulation. This study supports further examination of ferruginol as a potential agent for both the prevention and treatment of prostate cancer.
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http://dx.doi.org/10.1016/j.biochi.2008.01.011DOI Listing
June 2008

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

J Mol Histol 2008 Apr 7;39(2):201-8. Epub 2007 Nov 7.

Department of Endodontics, Bauru Dental School, University of Sao Paulo, Bauru, SP, Brazil.

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.
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http://dx.doi.org/10.1007/s10735-007-9152-zDOI Listing
April 2008

A possible anti-proliferative and anti-metastatic effect of irradiated riboflavin in solid tumours.

Cancer Lett 2007 Dec;258(1):126-34

Departamento de Bioquímica, Universidade Estadual de Campinas, Brazil.

Riboflavin is a potent photosensitizer as well as part of the vitamin B complex. Recently we demonstrated that the products generated by irradiation of riboflavin have potential as anti-leukaemic therapy. The possible action, however, of the riboflavin photoproducts in solid cancers has not been addressed. Hence, we investigated the effects of irradiated riboflavin on androgen-independent human prostate cancer cells (PC3), a known model for solid tumour cells with an exceptional resistance to therapy. Our results show that riboflavin photoproducts are cytotoxic to these cells in a FasL-Fas-dependent manner. Furthermore, irradiated riboflavin inhibited matrix-degrading proteases, caused downregulation of VEGF and upregulation of TIMP1 suggesting anti-metastatic potential. Together, these results show that the anti-neoplastic action of riboflavin photoproducts is not limited to haematological malignancies, warranting clinical studies in solid tumours.
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http://dx.doi.org/10.1016/j.canlet.2007.08.024DOI Listing
December 2007
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