In Vitro and In Vivo Characterization of PLLA-316L Stainless Steel Electromechanical Devices for Bone Tissue Engineering-A Preliminary Study.

Bone injuries represent a major social and financial impairment, commonly requiring surgical intervention due to a limited healing capacity of the tissue, particularly regarding critical-sized defects and non-union fractures. Regenerative medicine with the application of bone implants has been developing in the past decades towards the manufacturing of appropriate devices. This work intended to evaluate medical 316L stainless steel (SS)-based devices covered by a polymer poly (L-lactic acid) (PLLA) coating for bone lesion mechanical and functional support. SS316L devices were subjected to a previously described silanization process, following a three-layer PLLA film coating. Devices were further characterized and evaluated towards their cytocompatibility and osteogenic potential using human dental pulp stem cells, and biocompatibility via subcutaneous implantation in a rat animal model. Results demonstrated PLLA-SS316L devices to present superior in vitro and in vivo outcomes and suggested the PLLA coating to provide osteo-inductive properties to the device. Overall, this work represents a preliminary study on PLLA-SS316L devices' potential towards bone tissue regenerative techniques, showing promising outcomes for bone lesion support.

Alginate-nanohydroxyapatite hydrogel system: Optimizing the formulation for enhanced bone regeneration.

Ceramic/polymer-based biocomposites have emerged as potential biomaterials to fill, replace, repair or regenerate injured or diseased bone, due to their outstanding features in terms of biocompatibility, bioactivity, injectability, and biodegradability. However, these properties can be dependent on the amount of ceramic component present in the polymer-based composite. Therefore, in the present study, the influence of nanohydroxyapatite content (30 to 70 wt%) on alginate-based hydrogels was studied in order to evaluate the best formulation for maximizing bone tissue regeneration. The composite system was characterized in terms of physic-chemical properties and biological response, with in vitro cytocompatibility assessment with human osteoblastic cells and ex vivo functional evaluation in embryonic chick segmental bone defects. The main morphological characteristics of the alginate network were not affected by the addition of nanohydroxyapatite. However, physic-chemical features, like water-swelling rate, stability at extreme pH values, apatite formation, and Ca2+ release were nanoHA dose-dependent. Within in vitro cytocompatibility assays it was observed that hydrogels with nanoHA 30% content enhanced osteoblastic cells proliferation and expression of osteogenic transcription factors, while those with higher concentrations (50 and 70%) decreased the osteogenic cell response. Ex vivo data underlined the in vitro findings, revealing an enhanced collagenous deposition, trabecular bone formation and matrix mineralization with Alg-nanoHA30 composition, while compositions with higher nanoHA content induced a diminished bone tissue response. The outcomes of this study indicate that nanohydroxyapatite concentration plays a major role in physic-chemical properties and biological response of the composite system and the optimization of the components ratio must be met to maximize bone tissue regeneration.

Potential anti-cancer and anti-Candida activity of Zn-derived foams.

Zinc (Zn)-derived foams have been prepared from an alkaline electrolyte solution by galvanostatic electrodeposition under different conditions. A detailed physico-chemical characterization was performed by Raman spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). A pioneer application of these foams in medical implant-related applications was investigated. The in vitro behaviour of these Zn-derived foams in simulated physiological conditions was studied. The results revealed that the presence of zinc oxide was important enough to change the in vitro behaviour of these materials. The potential of these Zn-derived foams in inhibiting bone cancer cell proliferation - osteoscarcoma cells - and important pathogenic fungi responsible for implant-related infections -Candida albicans- was examined. Furthermore, the foams were evaluated for cytocompatibility with normal human osteoblasts. The results obtained allowed us to conclude that Zn-derived foams have an interesting potential for anti-cancer and anti-Candida activity, targeted for bone-related implant applications, suggesting that this novel material may have potential for further clinical studies.

"Chair Stand Test" as Simple Tool for Sarcopenia Screening in Elderly Women.

OBJECTIVES: To investigate the association between sarcopenia and "chair stand test" performance, and evaluate this test as a screening tool for sarcopenia in community-dwelling elderly women. DESIGN: Cross-sectional Survey. PARTICIPANTS: 173 female individuals, aged ≥ 60 years and living in the urban area of the municipality of Lafaiete Coutinho, Bahia's inland, Brazil. MEASUREMENTS: The association between sarcopenia (defined by muscle mass, strength and/or performance loss) and performance in the "chair stand test" was tested by binary logistic regression technique. The ROC curve parameters were used to evaluate the diagnostic power of the test in sarcopenia screening. The significance level was set at 5 %. RESULTS: The model showed that the time spent for the "chair stand test" was positively associated (OR = 1.08; 95% CI = 1.01 - 1.16, p = 0.024) to sarcopenia, indicating that, for each 1 second increment in the test performance, the sarcopenia's probability increased by 8% in elderly women. The cut-off point that showed the best balance between sensitivity and specificity was 13 seconds. CONCLUSION: The performance of "chair stand test" showed predictive ability for sarcopenia, being an effective and simple screening tool for sarcopenia in elderly women. This test could be used for screening sarcopenic elderly women, allowing early interventions.

Anti-sessile bacterial and cytocompatibility properties of CHX-loaded nanohydroxyapatite.

Nanohydroxyapatite possesses exceptional biocompatibility and bioactivity regarding bone cells and tissues, justifying its use as a coating material or as a bone substitute. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. Surface functionalization with antimicrobials is a promising strategy to reduce the likelihood of bacterial infestation and colonization on medical devices. Chlorhexidine digluconate is a common and effective antimicrobial agent used for a wide range of medical applications. The purpose of this work was the development of a nanoHA biomaterial loaded with CHX to prevent surface bacterial accumulation and, simultaneously, with good cytocompatibility, for application in the medical field. CHX (5-1500 mg/L) was loaded onto nanoHA discs and the materials were evaluated for CHX adsorption and release profile, physic-chemical features, antibacterial activity against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, and cytocompatibility toward L929 fibroblasts. Results showed that the adsorption of CHX on nanoHA surface occurred by electrostatic interactions between the cationic group of CHX and the phosphate group of nanoHA. The release of CHX from CHX-loaded nanoHA showed a fast initial rate followed by a slower kinetics release, due to constraints caused by dilution and diffusion-limiting processes. NanoHA.50 to nanoHA.1500 showed strong anti-sessile activity, inhibiting bacterial adhesion and the biofilm formation. CHX-nanoHA caused a dose- and time-dependent inhibitory effect on the proliferation of fibroblasts for nanoHA.100 to nanoHA.1500. Cellular behavior on nanoHA.5 and nanoHA.50 was similar to control. Therefore, CHX-loaded nanoHA surfaces appear as a promising alternative to prevention of devices-related infections.

The benefit of a human bone marrow stem cells concentrate in addition to an inorganic scaffold for bone regeneration: an in vitro study.

Background. This work compares the osteoblastic behaviour of a bone marrow (BM) aspirate and a prepared BM concentrate of nucleated cells associated with a glass reinforced hydroxyapatite composite (GRHC) in a microporous pellet formulation. Methods. BM aspirate (30 mL) was collected during 3 orthopedic surgical procedures, and a concentration system was used to achieve 3 rapid preparations of a concentrate of nucleated cells (3 mL) from the BM aspirates. The BM aspirates (53% cell viability; 2.7 × 10(6) nucleated cell/mL) and the BM concentrates (76% cell viability; 2 × 10(7) nucleated cell/mL) were cultured over glass reinforced hydroxyapatite pellets, at the same volume/mass ratio, for 30 days. Cultures performed in standard tissue culture plates were used as control. Results. The colonized BM concentrate/material constructs exhibited a representative osteoblastic proliferation/differentiation pathway, evidenced by a high alkaline phosphatase (ALP) activity, expression of collagen type 1, ALP, BMP-2, M-CSF, RANKL, and OPG, and formation of a calcium phosphate mineralized matrix. A clear improved behaviour was noticed compared to the BM aspirate/material constructs. Conclusions. The results suggest the benefit of using an autologous BM concentrate/material construct in the clinical setting, in bone regeneration applications.

The Osteogenic Priming of Mesenchymal Stem Cells is Impaired in Experimental Diabetes.

Diabetes mellitus encompasses a group of metabolic conditions embracing the dysfunction and failure of various tissues and organs, including bone. Sustained bone alterations seem to result from anabolic, rather than catabolic processes, and suggest a decreased osteoblastic recruitment and activity. Current knowledge on the cellular and molecular mechanisms were provided by studies performed with osteogenic populations cultured in diabetic-simulated conditions, and osteogenic-induced precursor populations harvested from diabetic animals, sustaining an impaired cellular behavior in terms of osteogenic responsiveness and function. However, the reasons leaning to this impairment remain essentially unknown, as the priming capability and functionality of undifferentiated precursors, developed within the diabetic environment, have not been addressed. Accordingly, this work aims to evaluate the functionality and osteogenic priming capability of bone marrow-derived mesenchymal stem cells (MSCs), harvested from animals with experimental diabetes, and grown in the absence of any given differentiation factor. MSCs developed within a diabetic microenvironment displayed an impaired behavior, with diminished cell viability and proliferation, altered cytoskeleton organization, impaired osteogenic priming, and increased adipogenic activation. Further, the osteogenic induction of diabetic MSCs resulted in an impaired osteogenic commitment. The modified cell phenotype may be related, at least in part, with altered activity of ERK WNT and p38 signaling pathways in diabetic-derived cultures. Specific strategies, aiming the modulation of the verified hindrances, may be of therapeutic value to enhance the functionality of diabetic MSCs and sustain an improved outcome in the metabolism and regeneration of the bone tissue in diabetic conditions.

Sarcosine oxidase composite screen-printed electrode for sarcosine determination in biological samples.

As the prostate cancer (PCa) progresses, sarcosine levels increase both in tumor cells and urine samples, suggesting that this metabolite measurements can help in the creation of non-invasive diagnostic methods for this disease. In this work, a biosensor device was developed for the quantification of sarcosine via electrochemical detection of H2O2 (at 0.6V) generated from the catalyzed oxidation of sarcosine. The detection was carried out after the modification of carbon screen printed electrodes (SPEs) by immobilization of sarcosine oxidase (SOX) on the electrode surface. The strategies used herein included the activation of the carbon films by an electrochemical step and the formation of an NHS/EDAC layer to bond the enzyme to the electrode, the use of metallic or semiconductor nanoparticles layer previously or during the enzyme immobilization. In order to improve the sensor stability and selectivity a polymeric layer with extra enzyme content was further added. The proposed methodology for the detection of sarcosine allowed obtaining a limit of detection (LOD) of 16nM, using a linear concentration range between 10 and 100nM. The biosensor was successfully applied to the analysis of sarcosine in urine samples.

The biomaterial-mediated healing of critical size bone defects in the ovariectomized rat.

UNLABELLED: This study demonstrated an impaired biomaterial-mediated bone regeneration in a critical sized calvarial defect established within an ovariectomized rat model. Histological and microtomographic evidences were supported by an impaired osteoblastic gene expression and altered expression of estrogen receptors and adipogenic markers. INTRODUCTION: This work aims to address the bone regeneration process in the ovariectomized rat model, by assessing a calvarial critical size defect implanted with a biocompatible bovine bone mineral graft. METHODS: Animals were randomly divided into two groups: Ovx (bilateral ovariectomy) and Sham (control surgery). Following 8 weeks, all animals were submitted to a surgical bicortical craniotomy (5-mm circular critical size defect), which was filled with a biocompatible mineral graft. Animals were euthanized at 1, 3, and 6 months following graft implantation (n = 10), and results on the orthotopic bone regeneration process were blindly evaluated by radiographic, microtomographic, histological, histomorphometric, and gene expression techniques. RESULTS: In the attained model, in both Sham and Ovx groups, the bone regenerative process was found to occur in a slow-paced manner. Likewise, a qualitative evaluation of the microtomographic and histological analysis, as well as quantitative data from histomorphometric indexes, revealed reduced bone regeneration in Ovx animals, at the assayed time points. Significant differences were attained at the 3 and 6 months. Gene expression analysis revealed a reduced expression of osteoblastic-related genes and an altered expression of estrogen receptors and adipogenic markers, within the regenerating bone of Ovx animals. CONCLUSIONS: Due to the similarities between the osteoporotic animal model and the human condition of postmenopausal osteoporosis, it might be relevant to consider the potential clinical implication of the osteoporotic condition in the biomaterial-mediated bone tissue healing/regeneration process.

Response of monocultured and co-cultured human microvascular endothelial cells and mesenchymal stem cells to macroporous granules of nanostructured-hydroxyapatite agglomerates.

Recent studies have shown that hydroxyapatite (HA) nanocrystalline have better functional properties that are important to create suitable local conditions for bone formation, when implanted in an osseous environment. Bone formation depends on several complex processes, including a tight communication between endothelial cells and osteoblasts and mesenchymal stem cells. This study examined the interaction between human dermal microvascular endothelial cells (HDMEC) and human mesenchymal stem cells (HMSC), in monoculture and co-culture on,macroporous granules of nanostructured-hydroxyapatite agglomerates. Cell viability/proliferation was assessed through MTT and DNA quantification assays. CLSM and SEM observations allow the study of cell morphology and growth pattern of cells. The angiogenic and osteogenic genes expression were studied using real time PCR and cell differentiation was assessed by ALP activity and matrix mineralization assays. Matrigel tube-like formation assay was also used. Increased expression levels of genes related with osteogenesis and angiogenesis was evident. The osteoblastic phenotype was clearly promoted, as evidenced by the over-expression of osteoblastic genes, increased ALP activity and matrix mineralization. The work clearly demonstrated that the nanostructured-HA granules were able to support cell type's survival, proliferation and individual functionality in a monoculture and co-culture system, for 21 days. HMSC seeded on the granules were able to differentiate into osteoblastic phenotype. The results achieved suggest that nano-structured HA granules may be considered promising implants for bone regeneration and tissue engineering application, in which the granules can be pre-seeded with these two types of autologous cells, before bone graft implant.

Acrylic formulations containing bioactive and biodegradable fillers to be used as bone cements: properties and biocompatibility assessment.

The solid phase of bioactive self-curing acrylic cements was modified by different biodegradable fillers such as poly(3-hydroxybutyrate) (PHB) and its copolymer with hydroxyvalerate (PHBV). The addition of the biodegradable fillers made the cement partially degradable, which is important to allow new bone replacement and ingrowth. The thermal analysis, crystallinity, curing parameters, mechanical properties, degradation and cellular tests were studied in order to characterize the cement performance. Within this context it was verified that the incorporation of the PHBV polymer made the cement more resistant, reaching values within the range reported for typical PMMA bone cements. The results also showed that the cement filled with PHBV took up more water than the cement with PHB after 60 days, for all studied formulations. Regarding the osteoblastic cytocompatibility assessment, the inclusion of the PHBV greatly improved the biological response in both cements filled with the silicate or the borate glass, compared to the inclusion of the PHB. The importance of this novel approach resides on the combination of the properties of the cements components and the possibility of allowing bone regeneration, improving the interfaces with both the prosthesis and the bone, and leading to a new material with suitable performance for application as bone cement.

Inhibition of human in vitro osteoclastogenesis by Equisetum arvense.

OBJECTIVES: Equisetum arvense has long been used in traditional medicines to treat different disorders, including bone pathologies. In this study a hydromethanolic extract of E. arvense was assessed for its effects on human osteoclastogenesis. MATERIALS AND METHODS: Osteoclast precursors were maintained in non-stimulated and stimulated (presence of M-CSF and RANKL) conditions, or in co-cultures with osteoblasts. Cell cultures were treated with 0.00016-0.5 mg/ml of a hydromethanolic E. arvense extract. RESULTS: The extract did not affect spontaneous osteoclastogenesis. In osteoclast precursors committed to osteoclastogenesis (stimulated or co-cultured with osteoblasts), E. arvense caused dose-dependent inhibitory effect that became statistically significant at concentrations ≥0.004 mg/ml. This was observed using different osteoclast differentiation and activation markers. Cell response was associated with changes in relative contribution of MEK and NFkB signalling pathways, as well as PGE2 production. As there were differences in the response of osteoclast precursors maintained in the presence of inductive factors, or co-cultured with osteoblastic cells, it seems that E. arvense extract had the ability to modulate osteoclastogenesis, either by acting directly on osteoclast precursor cells, and/or via osteoblasts. CONCLUSIONS: Equisetum appeared to have a negative effect on human osteoclastogenesis, which is in line with its putative beneficial role in pathophysiological conditions associated with increased osteoclastic activity, and might suggest potential utility for treatment with bone regeneration strategies.

Micropatterned silica thin films with nanohydroxyapatite micro-aggregates for guided tissue regeneration.

UNLABELLED: Surface modification of biomaterials has been shown to improve the biological response to dental implants. The ability to create a controlled micro-texture on the implant via additive surface modification techniques with bioactive nanohydroxyapatite (nanoHA) may positively influence guided tissue regeneration. OBJECTIVE: The main goal of this study was to produce micro-fabricated SiO(2) surfaces modified with nanohydroxyapatite particles and to characterize their influence on the biological response of Human Dental-Pulp Mesenchymal Stem Cells (hDP-MSCs) and Streptococcus mutans. MATERIALS AND METHODS: A combined methodology of sol-gel and soft-lithography was used to produce micropatterned SiO(2) thin films with different percentages of nanoHA micro-aggregates. The surfaces were characterized by SEM/EDS, FT-IR/ATR, AFM, XPS quantitative elemental percentage and contact angle measurements. Biological characterization was performed using hDP-MSCs cultures, while Streptococcus mutans was the selected microorganism to evaluate the bacterial adhesion on the thin films. RESULTS: Micropatterned SiO(2) surfaces with 0%, 1% and 5% of nanoHA micro-aggregates were successfully produced using a combination of sol-gel and soft-lithography. These surfaces controlled the biological response, triggering alignment and oriented proliferation of hDP-MSCs and significant differences in the adhesion of S. mutans to the different surfaces. SIGNIFICANCE: The micropatterned surfaces exhibited biocompatible behavior that induced an oriented adhesion and proliferation of hDP-MSCs while SiO(2) presented low bacterial adhesion. These results show that the combination of sol-gel with soft-lithography is a good approach to create micropatterned surfaces with bioactive nanoparticles for guided tissue regeneration.

Reciprocal induction of human dermal microvascular endothelial cells and human mesenchymal stem cells: time-dependent profile in a co-culture system.

OBJECTIVES: Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration. For these reasons, the aim of this work was to develop a co-culture system to study in detail any time-dependent interactions between human mesenchymal stem cells (HMSC) and human dermal microvascular endothelial cells (HDMEC), co-cultured in a 2D system, for 35 days. MATERIALS AND METHODS: HMSC and HDMEC were co-cultured at a ratio of 1:4, respectively. Single-cell cultures were used as controls. Cell viability/proliferation was assessed using MTT, DNA quantification and calcein-AM assays. Cell morphology was monitored using confocal microscopy, and real time PCR was performed. Alkaline phosphatase activity and histochemical staining were evaluated. Matrix mineralization assays were also performed. RESULTS: Cells were able to grow in characteristic patterns maintaining their viability and phenotype expression throughout culture time, compared to HMSC and HDMEC monocultures. HMSC differentiation seemed to be enhanced in the co-culture conditions, since it was observed an over expression of osteogenesis-related genes, and of ALP activity. Furthermore, presence of calcium phosphate deposits was also confirmed. CONCLUSIONS: This work reports in detail the interactions between HMSC and HDMEC in a long-term co-culture 2D system. Endothelial and mesenchymal stem cells cultured in the present co-culture conditions ensured proliferation and phenotype differentiation of cell types, osteogenesis stimulation and over-expression of angiogenesis-related genes, in the same culture system. It is believed that the present work can lead to significant developments for bone tissue regeneration and cell biology studies.

Induced osteoclastogenesis by fluoroquinolones in unstimulated and stimulated human osteoclast precursor cells.

Fluoroquinolones (FQs) are a class of antibiotics with a broad spectrum of activity, known to disturb bone metabolism. The aim of this work was to characterize the cellular and molecular effects of five FQs (ofloxacin, norfloxacin, ciprofloxacin, levofloxacin and moxifloxacin) in unstimulated and stimulated human osteoclast precursors. Peripheral blood mononuclear cells (PBMC) were cultured in the absence (unstimulated) or in the presence of osteoclastogenic factors (M-CSF and RANKL, stimulated), and were treated with FQs (0.3×10(-9)-10(-3) M), for 21 days. In unstimulated PBMC cultures, FQs (excepting moxifloxacin) exhibited a high osteoclastogenic potential, as shown by a significant increase in the expression of osteoclastic genes, TRAP activity and, specially, number of TRAP-positive multinucleated cells and calcium phosphate resorbing ability, suggesting the presence of mature and functional osteoclasts. Norfloxacin and levofloxacin induced the higher effect, followed by ciprofloxacin and ofloxacin. A decrease on apoptosis and an increase on M-CSF expression might have a possible contribution in the observed cellular behavior. In stimulated PBMC cultures, FQs further increase the osteoclastogenic response induced by M-CSF and RANKL (except ofloxacin). However, the osteoclastogenic response was much lower than that observed in unstimulated PBMC cultures. Both in unstimulated and stimulated PBMC cultures, for most of the FQs, the osteoclastogenic effects were observed in a wide range of concentrations, representative of plasmatic and tissue levels attained in several clinical settings. The various FQs differed on the stimulatory concentration range, the extent of the induced osteoclastogenic response and, also, on the dose- and time-dependent profile. Nevertheless, at high concentrations all the FQs seemed to elicit an increase on apoptosis. Additionally, some differences were noted in the intracellular signaling pathways tested, namely NFkB, MEK and PGE2 production. Results suggest that, considering the inter-individual variability of the FQs pharmacokinetics, the detailed biological profile of each FQ on bone cells is of utmost importance to clarify the effects of these compounds on bone metabolism.

Spontaneous and induced osteoclastogenic behaviour of human peripheral blood mononuclear cells and their CD14(+) and CD14(-) cell fractions.

OBJECTIVES: Osteoclasts are descended from the CD14(+) monocyte/macrophage lineage, but influence of other haematopoietic cells on osteoclastic commitment of their precursors has remained poorly understood. In this study, osteoclastogenic behaviour of peripheral blood mononuclear cells (PBMC) and their CD14(+) and CD14(-) subpopulations has been accessed, in the absence or presence of M-CSF and RANKL. MATERIALS AND METHODS: Cell cultures were characterized for presence of actin rings and vitronectin and calcitonin receptors, TRAP activity and calcium phosphate resorbing activity, expression of osteoclast-related genes and secretion of M-CSF and RANKL. RESULTS: In the absence of growth factors, PBMC and CD14(+) cultures had some degree of cell survival, and some spontaneous osteoclastogenesis was observed, only on cultures of the former. Supplementation with M-CSF and RANKL significantly increased osteoclastogenic behaviour of cell cultures, particularly CD14(+) cell cultures. Nevertheless, PBMC derived a higher degree of osteoclastogenesis, either as absolute values or after normalization by protein content. It was observed that unlike CD14(+) cells, PBMC were able to express M-CSF and RANKL, which increased following growth factor treatment. Also, expression of TNF-α, GM-CSF, IL-1ß, IL-6 and IL-17 was higher in PBMC cultures. Finally, CD14(-) cultures exhibited limited cell survival and did not reveal any osteoclast features. CONCLUSIONS: Results show that although osteoclastic precursors reside in the CD14(+) cell subpopulation, other populations (such as CD14(-) cells) derived from PBMC, have the ability to modulate osteoclastogenesis positively.

Paracrine-mediated differentiation and activation of human haematopoietic osteoclast precursor cells by skin and gingival fibroblasts.

OBJECTIVE: Fibroblasts appear to modulate osteoclastogenesis, but their precise role in this process remains unclear. In this work, paracrine-mediated osteoclastogenic potential of different human fibroblasts was assessed. MATERIALS AND METHODS: Fibroblast-conditioned media (CM) from foetal skin (CM1), adult skin (CM2) and adult gingiva (CM3) were used to promote osteoclastogenesis of osteoclast precursor cells. Cultures supplemented with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) were used as controls. RESULTS: All fibroblast cultures expressed FSP-1, M-CSF and RANKL and produced osteoprotegerin (OPG); gingival fibroblasts presented lowest expression of osteoclastogenic genes and higher production of OPG. All fibroblast CM were able to induce osteoclastogenesis. CM1 showed behaviour similar to positive controls, and slightly higher osteoclastogenic potential than CM, from adult ones. Gingival fibroblasts revealed lowest osteoclastogenic ability. Presence of anti-MCSF or anti-RANKL partially inhibited osteoclastogenesis promoted by CM, although the former antibody revealed higher inhibitory response. Differences among the osteoclastogenic effect of CM were noted, mainly in expression of genes involved in differentiation and activation of osteoclast precursor cells, c-myc and c-src, and less regarding functional related parameters. CONCLUSIONS: Fibroblasts are able to induce osteoclastogenesis by paracrine mechanisms, and age and anatomical location affect this ability. Other factors produced by fibroblasts, in addition to M-CSF and RANKL, appear to contribute to observed osteoclastogenic potential.

Silicate and borate glasses as composite fillers: a bioactivity and biocompatibility study.

Composites filled with a silicate glass (CSi) and a new borate glass (CB) were developed and compared in terms of their in vitro behaviour both in acellular and cellular media. Acellular tests were carried out in SBF and the composites were characterized by SEM-EDS, XRD and ICP. Biocompatibility studies were investigated by in vitro cell culture with MG-63 osteoblast-like and human bone marrow cells. The growth of spherical calcium phosphate aggregates was observed in acellular medium on all composite surfaces indicating that these materials became potentially bioactive. The biological assessment resulted in a dissimilar behavior of the composites. The CSi demonstrated an inductive effect on the proliferation of cells. The cells showed a normal morphology and high growth rate when compared to standard culture plates. Contrarily, inhibition of cell proliferation occurred in the CB probably due to its high degradation rate, leading to high B and Mg ionic concentration in the cell culture medium.

Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

Although in the past little attention has been paid to the influence of osteosarcoma cells in osteoclast function, recent studies suggest a close relationship between osteosarcoma aggressiveness and osteoclastic activity. The present study addresses the paracrine effects of MG63 cells, a human osteosarcoma-derived cell line, on the differentiation of peripheral blood osteoclast precursor cells (PBMC). PBMC were cultured for 21 days in the presence of conditioned media from MG63 cell cultures (CM) collected at 48 h (CM_MG1), 7 days (CM_MG2) and 14 days (CM_MG3). MG63 cell cultures displayed the expression of ALP and BMP-2 and, also, the osteoclastogenic genes M-CSF and RANKL, although with a low expression of RANKL. PBMC cultures supplemented with CM presented an evident osteoclastogenic behavior, which was dependent on the culture period of the MG63 cells. The inductive effect appeared to be more relevant for the differentiation and activation genes, c-myc and c-src, and lower for genes associated with osteoclast function. In addition, PBMC cultures displayed increased functional parameters, including calcium phosphate resorbing activity. Assessment of the PBMC cultures in the presence of U0126, PDTC, and indomethacin suggested that in addition to MEK and NFkB pathways, other signaling mechanisms, probably not involving RANKL/RANK interaction, might be activated in the presence of conditioned medium from MG63. In conclusion, MG63 cell line appears to induce a significant paracrine-mediated osteoclastogenic response. Understanding the mechanisms underlying the interaction of osteosarcoma cells and osteoclasts may contribute to the development of new potential approaches in the treatment of such bone metabolic diseases.

Mastocytosis: oral implications of a rare disease.

Mastocytosis encompasses a group of rare clinical entities, which are characterized by an abnormal growth and, usually, low accumulation of clonal and morphologically abnormal mast cells (MCs), within one or more organs. Clinical presentations are quite variable and symptoms are usually related to the release of mast cell mediators, tissue infiltration by MC (usually in the aggressive categories of the disease), or both. Mast cells are hematopoietic-derived cells that reach phenotypic maturity in the mucosa and peripheral connective tissues. These cells play an active role both on immunologic and non-immunologic processes. Within the oral cavity, MCs reside in the connective tissues, in physiologic conditions, and their number is elevated in pathologic situations resulting from immunoinflammatory processes, such as pulpal inflammation and periodontal disease. As MCs influence so many phenomena within the oral cavity, mastocytosis may manifest itself in the oral tissues. Patients with mastocytosis should be put under special care by dental professionals, in what concerns not only general patient management, but also drug prescription, as they are particularly prone to anaphylaxis and other peri and post-operative complications. Several allergens or mast cell activation triggers such as local anesthetics, zinc oxide, eugenol, penicilins, metals and oral hygiene products are frequently administered or prescribed by dentists. Patients with mastocytosis may also require stress management, during dental consultation. This review aims to briefly summarize the potential ways in which mast cell disease may affect the oral cavity and the dental management of mastocytosis affected patients.