Green tea and hyaluronic acid gel enhance fibroblast activation and improves the gingival healing post-third molar extraction.

This study evaluates the effects of a green tea (Camellia sinensis) and hyaluronic acid gel on fibroblast activity and alveolar bone repair following third molar extractions. By examining the gene expression related to cell survival, proliferation, and angiogenesis, the study bridges in vitro findings with clinical outcomes in a split-mouth randomized trial. Human fibroblasts were exposed to the treatment gel, analysing gene expression through RT-qPCR. Twenty participants undergoing bilateral third molar extractions received the test gel on one side and a placebo on the other. Assessments included patient-reported outcomes, professional evaluations, and radiographic analyses at multiple postoperative intervals. The test gel significantly enhanced AKT, CDKs, and VEGF gene expressions, indicating a positive effect on angiogenesis and cell proliferation. Clinically, it resulted in reduced exudate, swelling, and secondary interventions, with radiographs showing improved alveolar bone density after 90 days. The green tea and hyaluronic acid gel significantly improves soft tissue and bone healing post-extraction, offering a promising adjunctive therapy for enhancing postoperative recovery. This gel represents a novel adjuvant treatment option for facilitating improved healing outcomes after third molar extractions, highlighting its potential utility in clinical dental practice.

Development of cobalt (Co)-doped monetites for bone regeneration.

Cobalt-doped monetite powders were synthesized by coprecipitation method under a cobalt nominal content between 2 and 20 mol % of total cation. Structural characterization of samples was performed by using X-ray diffraction (XRD), Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy. XRD results indicated that the Co-doped samples exhibited a monetite single-phase with the cell parameters and crystallite size dependent on the amount of substitutional element incorporated into the triclinic crystalline structure. Cell viability and adhesion assays using pre-osteoblastic cells showed there is no toxicity and the RTqPCR analysis showed significant differences in the expression for osteoblastic phenotype genes, showing a potential material for the bone regeneration.

Hypoxia modulates the phenotype of mechanically stressed endothelial cells responding to CoCr-enriched medium.

Given the importance of the endothelial cell phenotype in dental peri-implant healing processes, the aim of this study was to better assess the involvement of endothelial cells responding to cobalt-chromium (CoCr)-enriched medium. Biologically, cobalt is widely used molecule to induce chemical experimental hypoxia because it stabilizes hypoxia inducible factors (HIF1α). The aplication of hypoxia models provides better experimental condition to allow its impact on cellular metabolism, by looking for biochemical and molecular issues. Thus, this study looks for understaing whether CoCr-based materials are able to modulate endothelial cells considering the hypoxic effect prmoted by cobalt. Firstly, our data shows there is a siginificant effect on endothelial phenotype by modulating the expression of VEGF and eNOS genes, whith low requirement of genes related with proteasome intracellular complex. Importantly, the data were validated using classical chemical modulators of hypoxia signaling [chrysin (5,7-dihydroxyflavone) and Dimethyloxalylglycine (DMOG)] in functional assays. Altogether, these data validate the hypothesis that hipoxya is important to maintain the phenotype of endothelial cells, and it is properly interesting during the tissue regeneration surrounding implants and so compromising osseointegration process. Finally, it is important to mention that the cobalt released from CoCr devices might contribute with an sufficient microenvironment surrounding implanted devices and it paviments new roads looking for more bioactive surfaces of implantable materials in human health.

The Action of Angiocrine Molecules Sourced from Mechanotransduction-Related Endothelial Cell Partially Explain the Successful of Titanium in Osseointegration.

Since Branemark's findings, titanium-based alloys have been widely used in implantology. However, their success in dental implants is not known when considering the heterogenicity of housing cells surrounding the peri-implant microenvironment. Additionally, they are expected to recapitulate the physiological coupling between endothelial cells and osteoblasts during appositional bone growth during osseointegration. To investigate whether this crosstalk was happening in this context, we considered the mechanotransduction-related endothelial cell signaling underlying laminar shear stress (up to 3 days), and this angiocrine factor-enriched medium was harvested further to use exposing pre-osteoblasts (pOb) for up to 7 days in vitro. Two titanium surfaces were considered, as follows: double acid etching treatment (w_DAE) and machined surfaces (wo_DAE). These surfaces were used to conditionate the cell culture medium as recommended by ISO10993-5:2016, and this titanium-enriched medium was later used to expose ECs. First, our data showed that there is a difference between the surfaces in releasing Ti molecules to the medium, providing very dynamic surfaces, where the w_DAE was around 25% higher (4 ng/mL) in comparison to the wo_DAE (3 ng/mL). Importantly, the ECs took up some of this titanium content for up to 3 days in culture. However, when this conditioned medium was used to expose pOb for up to 7 days, considering the angiocrine factors released from ECs, the concentration of Ti was lesser than previously reported, reaching around 1 ng/mL and 2 ng/mL, respectively. Thereafter, pOb exposed to this angiocrine factor-enriched medium presented a significant difference when considering the mechanosignaling subjected to the ECs. Shear-stressed ECs showed adequate crosstalk with osteoblasts, stimulating the higher expression of the Runx2 gene and driving higher expressions of Alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin. Mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules also stimulated the higher expression of the Col3A1 gene in osteoblasts, which suggests it is a relevant protagonist during trabecular bone growth. In fact, we investigated ECM remodeling by first evaluating the expression of genes related to it, and our data showed a higher expression of matrix metalloproteinase (MMP) 2 and MMP9 in response to mechanosignaling-based angiocrine molecules, independent of considering w_DAE or the wo_DAE, and this profile reflected on the MMP2 and MMP9 activities evaluated via gelatin-based zymography. Complimentarily, the ECM remodeling seemed to be a very regulated mechanism in mature osteoblasts during the mineralization process once both TIMP metallopeptidase inhibitor 1 and 2 (TIMP1 and TIMP2, respectively) genes were significantly higher in response to mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules. Altogether, our data show the relevance of mechanosignaling in favoring ECs' release of bioactive factors peri-implant, which is responsible for creating an osteogenic microenvironment able to drive osteoblast differentiation and modulate ECM remodeling. Taking this into account, it seems that mechanotransduction-based angiocrine molecules explain the successful use of titanium during osseointegration.

Recombinant sugarcane cystatin CaneCPI-5 promotes osteogenic differentiation.

Cysteine proteases orchestrate bone remodeling, and are inhibited by cystatins. In reinforcing our hypothesis that exogenous and naturally obtained inhibitors of cysteine proteases (cystatins) act on bone remodeling, we decided to challenge osteoblasts with sugarcane-derived cystatin (CaneCPI-5) for up to 7 days. To this end, we investigated molecular issues related to the decisive, preliminary stages of osteoblast biology, such as adhesion, migration, proliferation, and differentiation. Our data showed that CaneCPI-5 negatively modulates both cofilin phosphorylation at Ser03, and the increase in cytoskeleton remodeling during the adhesion mechanism, possibly as a prerequisite to controlling cell proliferation and migration. This is mainly because CaneCPI-5 also caused the overexpression of the CDK2 gene, and greater migration of osteoblasts. Extracellular matrix remodeling was also evaluated in this study by investigating matrix metalloproteinase (MMP) activities. Our data showed that CaneCPI-5 overstimulates both MMP-2 and MMP-9 activities, and suggested that this cellular event could be related to osteoblast differentiation. Additionally, differentiation mechanisms were better evaluated by investigating Osterix and alkaline phosphatase (ALP) genes, and bone morphogenetic protein (BMP) signaling members. Altogether, our data showed that CaneCPI-5 can trigger biological mechanisms related to osteoblast differentiation, and broaden the perspectives for better exploring biotechnological approaches for bone disorders.

Cyclopamine targeting hedgehog modulates nuclear control of the osteoblast activity.

It is known that cellular events underlying the processes of bone maintenance, remodeling, and repair have their basis in the embryonic production of bone. Shh signaling is widely described developing important morphogenetic control in bone by modifying the activity of osteoblast. Furthermore, identifying whether it is associated with the modulation of nuclear control is very important to be the basis for further applications. Experimentally, osteoblasts were exposed with cyclopamine (CICLOP) considering up to 1 day and 7 days, here considered an acute and chronic responses respectively. Firstly, we have validated the osteogenic model in vitro by exposing the osteoblasts to classical differentiating solution up to 7 days to allow the analysis of alkaline phosphatase and mineralization. Conversely, our data shows that differentiating osteoblasts present higher activity of inflammasome-related genes, while Shh signaling members were lower, suggesting a negative feedback between them. Thereafter, to better know about the role of Shh signaling on this manner, functional assays using CICLOP (5 µM) were performed and the data validates the previously hypothesis that Shh represses inflammasome related genes activities. Altogether, our data supports the anti-inflammatory effect of Shh signaling by suppressing Tnfα, Tgfß and inflammasome related genes during osteoblast differentiation, and this comprehension might support the understanding the molecular and cellular mechanisms related in bone regeneration by reporting molecular-related osteoblast differentiation.

Preparation and characterization of novel as-cast Ti-Mo-Nb alloys for biomedical applications.

Ti and its alloys are the most used metallic biomaterials devices due to their excellent combination of chemical and mechanical properties, biocompatibility, and non-toxicity to the human body. However, the current alloys available still have several issues, such as cytotoxicity of Al and V and high elastic modulus values, compared to human bone. ß-type alloys, compared to α-type and (α + ß)-type Ti alloys, have lower elastic modulus and higher mechanical strength. Then, new biomedical ß-type alloys are being developed with non-cytotoxic alloying elements, such as Mo and Nb. Therefore, Ti-5Mo-xNb system alloys were prepared by argon arc melting. Chemical composition was evaluated by EDS analysis, and the density measurements were performed by Archimedes' method. The structure and microstructure of the alloys were obtained by X-ray diffraction and optical and scanning electron microscopy. Microhardness values were analyzed, and MTT and crystal violet tests were performed to assess their cytotoxicity. As the Nb concentration increases, the presence of the ß-Ti phase also grows, with the Ti-5Mo-30Nb alloy presenting a single ß-Ti phase. In contrast, the microhardness of the alloys decreases with the addition of Nb, except the Ti-5Mo-10Nb alloy, which has its microhardness increased probably due to the ω phase precipitation. Biological in-vitro tests showed that the alloys are not cytotoxic.

Metabolic effects of CoCr-enriched medium on shear-stressed endothelial cell and osteoblasts: A possible mechanism involving a hypoxic condition on bone healing.

Cobalt-chromium (CoCr)-based alloys have emerged as an interesting biomaterial within biomedical field, mainly considering their biocompatibility, resistance to corrosion and absence of magnetism; however, its effect on cell metabolism is barely known and this prompted us better evaluating whether CoCr-enriched medium affects the metabolism of both osteoblast and endothelial cells, and also if there is a coupling between them. This is also considered here the already-known effect of Cobalt (Co) as a hypoxic element. Firstly, discs of CoCr [subjecting (W) or not (Wo) to dual acid-etched (DAE)] were incubated into FBS-free cell culture medium up to 24 h (37 °C). This CoCr-enriched medium was further used to treat shear-stressed endothelial cells cultures up to 72 h. Thereafter, the conditioned medium containing metabolites of shear-stressed endothelial cells in response to CoCr-enriched medium was further used to subject osteoblast's cultures, when the samples were properly harvested to allow the analysis of the molecular issues. Our data shows that CoCr-enriched medium contains 1.5 ng-2.0 ng/mL of Co, which was captured by endothelial cells and osteoblasts in about 30% in amount and it seems modulate their metabolic pathways: shear-stressed endothelial cells expressed higher profile of HIF1α, VEGF and nNOS genes, while their global profile of protein carbonylation was lower than the control cultures, suggesting lower oxidative stress commitment. Additionally, osteoblasts responding to metabolites of CoCr-challenged endothelial cells show dynamic expression of marker genes in osteogenic differentiation, with alkaline phosphatase (ALP), osteocalcin, and bone sialoprotein (BSP) genes being significantly increased. Additionally, tensional shear-stress forces decrease the stimulus for ColA1gene expression in osteoblasts responding to endothelial cells metabolites, as well as modifying the extracellular matrix remodeling related genes. Analyzing the activities of matrix metalloproteinases (MMPs), the data shows that shear-stressed endothelial cells metabolites increase the activities of both MMP9 and MMP2 in osteoblasts. Altogether, our data shows for the first time that shear-stressed endothelial metabolites responding to CoCr discs contribute to osteogenic phenotype in vitro, and this predicts an active crosstalk between angiogenesis and osteogenesis during osseointegration of CoCr alloy and bone healing, maybe guided by the Co-induced hypoxic condition.

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

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.

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

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.

PI3K/AKT signaling drives titanium-induced angiogenic stimulus.

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.

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

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.

Bisphosphonate-based surface biofunctionalization improves titanium biocompatibility.

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 (TiO2) 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 TiO2 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 (TiO2 + 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.

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

A Correction to this paper has been published: https://doi.org/10.1007/s10856-020-06470-x.

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

It has been shown that 17ß-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-ß, 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-ß. 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.

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

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 Integrin-ß1, Fak, and Src. 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 (Runx2, Alp, Bsp). CONCLUSION: Altogether, our study found interesting osteogenic behavior of cells in response to alendronate and albumin surfaces, which indicates the need for in vivo analyses to better consider these surfaces before clinical trials within the biomedical field.

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

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.

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

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.

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

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.

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

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-1ß, 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.