Evaluation of the effects of the solution used for electrochemical dissolution of nickel-titanium endodontic files on dentine structure, microhardness and cell viability.

AIM: To evaluate the effects of the [NaF 12 g L-1  + NaCl 1 g L-1 ] solution used in the electrochemical dissolution process of fractured endodontic files, as well as its NiTi-containing product, on dentine hardness, topography and human fibroblast viability. METHODOLOGY: Sixty single-rooted human teeth were evaluated for dentine microhardness using the Vickers hardness test and the area and number of dentinal tubules by scanning electron microscopy. The samples were divided according to the dentine surface treatment: distilled water; 17% EDTA; [NaF 12 g L-1  + NaCl 1 g L-1 ]; and 17% EDTA + [NaF 12 g L-1  + NaCl 1 g L-1 ]. Thirty-six single-rooted human teeth were divided according to the irrigation protocol: Dulbecco's Modified Eagle's Medium + 10% foetal bovine serum; 5.25% NaOCl; [NaF 12 g L-1  + NaCl 1 g L-1 ]; and [NaF 12 g L-1  + NaCl 1 g L-1  + NiTi]. The extracts in contact with the apical foramen were used in the MTT assay to evaluate human fibroblast viability, with dilutions of 100%, 50%, 25% and 12.5%. Statistical tests used were paired t-tests, one-way anova, Tukey's test, Kruskal-Wallis test and Dunn's post-test. RESULTS: The [NaF 12 g L-1  + NaCl 1 g L-1 ] solution did not modify dentine microhardness or the average dentinal tubule area. However, EDTA induced changes in dentine structure and microhardness (P < 0.05). The [NaF 12 g L-1  + NaCl 1 g L-1 ] solution, and its NiTi-containing product had lower cytotoxicity than NaOCl at dilutions of 25% and 50% (P < 0.01). CONCLUSIONS: The [NaF 12 g L-1  + NaCl 1 g L-1 ] solution did not alter dentine microhardness or damage the dentine structure. It also demonstrated lower cytotoxicity than NaOCl.

Fine-mapping of 5q12.1-13.3 unveils new genetic contributors to caries.

Caries is a multifactorial disease and little is still known about the host genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified the interval 5q12.1-5q13.3 as linked to low caries susceptibility in Filipino families. Here we fine-mapped this region in order to identify genetic contributors to caries susceptibility. Four hundred and seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. DMFT scores and genotype data of 75 single-nucleotide polymorphisms were evaluated in the Filipino families with the Family-Based Association Test. For replication purposes, a total 1,467 independent subjects from five different populations were analyzed in a case-control format. In the Filipino cohort, statistically significant and borderline associations were found between low caries experience and four genes spanning 13 million base pairs (PART1, ZSWIM6, CCNB1, and BTF3). We were able to replicate these results in some of the populations studied. We detected PART1 and BTF3 expression in whole saliva, and the expression of BTF3 was associated with caries experience. Our results suggest BTF3 may have a functional role in protecting against caries.

MMP13 polymorphism decreases risk for dental caries.

Recent evidence suggests that genetic studies may contribute to a better understanding of individual susceptibility to caries. Matrix metalloproteinases (MMPs) and their tissue inhibitors have been suggested to be involved in the caries process. The purpose of this study was to determine if polymorphisms in MMP2 (rs243865), MMP9 (rs17576), MMP13 (rs2252070), and TIMP2 (rs7501477) were associated with caries. Eligible unrelated children and adolescents were evaluated using a cross-sectional design. Data on oral health habits was obtained through a questionnaire and caries data was collected by clinical examination. Genotyping of the selected polymorphisms was carried out by real-time PCR. Allele and genotype frequencies were compared between individuals with and without caries experience. Of 505 subjects, 212 were caries-free and most subjects (61.2%) had mixed dentition. Allele frequency of MMP2, MMP13 and TIMP2 was different between caries-affected and caries-free individuals, with significant association for MMP13 (p = 0.004). Mutant allele carriers for MMP13 demonstrated a significantly decreased risk for caries (OR = 0.538, 95% CI 0.313-0.926); this result remained significant after adjustment for candidate genes, type of dentition and dietary factors. Allelic and genotype frequencies of the polymorphism in MMP9 were similar in caries-affected and caries-free individuals. Genetic variations in MMP13 may contribute to individual differences in caries susceptibility. Our findings reinforce that susceptibility to caries results from gene-environment interactions.

A multiparametric assay to compare the cytotoxicity of endodontic sealers with primary human osteoblasts.

AIM: To compare the cytotoxicity of four endodontic sealers (Sealapex, Pulp Canal Sealer EWT, Real Seal and MTA Fillapex) either 1 or 7 days after mixing, when assessed through a multiparametric analysis employing human primary cells closely related to periapical tissues. METHODOLOGY: Extracts of each sealer were prepared following 24-h exposure to culture media, at either 24 h or 7 days after mixing. Primary human osteoblasts were exposed to extracts for 24 h, at 37 °C with 5% CO(2) , and cell viability was evaluated by a multiparametric assay assessing sequentially, on the same cells, mitochondrial activity (XTT), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). Results from each test and experimental time were compared by 2-way analysis of variance (anova). RESULTS: All endodontic sealers had strong cytotoxicity 24 h after mixing, according to all parameters evaluated. At a longer setting period (7 days), viability for Sealapex was significantly increased (P < 0.05) and Pulp Canal Sealer achieved levels of cytocompatibility similar to the control group. The anova indicated a general correlation between the cytotoxicity of the materials and the time after mixing, with some level of dependence on the cell viability assay employed. CONCLUSIONS: All materials had high cytotoxic levels for human primary cells, mostly on a time-dependent basis, as shown by three different cell viability tests.

Cytocompatibility of the ready-to-use bioceramic putty repair cement iRoot BP Plus with primary human osteoblasts.

AIM: To verify the in vitro cytocompatibility of iRoot BP Plus (iRoot) and to compare it with White ProRoot MTA (MTA). METHODOLOGY: Thirty-six human maxillary incisor root canals were prepared using a step-back flaring technique. The apical 3 mm was resected perpendicular to the long axis at the roots, and root-end cavities were prepared with the aid of an ultrasonic device plus a diamond retrotip with continuous irrigation using water, producing standardized preparations. After that, the root-end cavities were filled with iRoot or MTA, and each root was exposed to cell culture media for 24 or 48 h. Human osteoblast cells were exposed to the extracts thus obtained, and a multiparametric cell viability assay was performed, evaluating mitochondrial activity, membrane integrity and cell density. The results were analysed by one-way analysis of variance, complemented with the Duncan post-test (P < 0.05). RESULTS: Cells exposed to MTA revealed a cytocompatibility pattern similar to the untreated cells (negative control), at both experimental times (P > 0.05). iRoot, however, promoted a significantly poorer viability than MTA and the control, after 48 h of exposure (P < 0.001). Nevertheless, iRoot did not induce critical cytotoxic effects because cell viability remained higher than 70% of the control group in most tests performed. CONCLUSION: iRoot and MTA were biocompatible and did not induce critical cytotoxic effects.

Impact of bisphosphonate therapy on dental implant outcomes: An overview of systematic review evidence.

The purpose of this overview was to assess the methods, quality, and outcomes of systematic reviews conducted to evaluate the impact of bisphosphonates on dental implants and the risk of developing bisphosphonate-related osteonecrosis of the jaw after dental implant surgery. An electronic search without date or language restriction was performed in the PubMed/MEDLINE, Cochrane CENTRAL, Web of Science, and LILACS databases (to January 2018). Eligibility criteria included systematic reviews that evaluated the impact of bisphosphonates on implant outcomes. The quality assessment of the included reviews was done using AMSTAR 2 guidelines. The protocol of this overview was registered in PROSPERO (CRD42018089617). The search and selection process yielded seven reviews, published between 2009 and 2017. None of the systematic reviews included in this study obtained the maximum score in the quality assessment. The scientific evidence available demonstrates that patients with a history of bisphosphonate use do not present a higher risk of dental implant failure or marginal bone loss compared to patients who have not used bisphosphonates. The literature also suggests that patients who undergo surgical trauma during the installation of dental implants may be more susceptible to bisphosphonate-related osteonecrosis of the jaw.

The two faces of titanium dioxide nanoparticles bio-camouflage in 3D bone spheroids.

Titanium (Ti) and its alloys are widely used in dental implants and hip-prostheses due to their excellent biocompatibility. Growing evidence support that surface degradation due to corrosion and wear processes, contribute to implant failure, since the release of metallic ions and wear particles generate local tissue reactions (peri-implant inflammatory reactions). The generated ions and wear debris (particles at the micron and nanoscale) stay, in a first moment, at the interface implant-bone. However, depending on their size, they can enter blood circulation possibly contributing to systemic reactions and toxicities. Most of the nanotoxicological studies with titanium dioxide nanoparticles (TiO2 NPs) use conventional two-dimensional cell culture monolayers to explore macrophage and monocyte activation, where limited information regarding bone cells is available. Recently three-dimensional models have been gaining prominence since they present a greater anatomical and physiological relevance. Taking this into consideration, in this work we developed a human osteoblast-like spheroid model, which closely mimics bone cell-cell interactions, providing a more realistic scenario for nanotoxicological studies. The treatment of spheroids with different concentrations of TiO2 NPs during 72 h did not change their viability significantly. Though, higher concentrations of TiO2 NPs influenced osteoblast cell cycle without interfering in their ability to differentiate and mineralize. For higher concentration of TiO2 NPs, collagen deposition and pro-inflammatory cytokine, chemokine and growth factor secretion (involved in osteolysis and bone homeostasis) increased. These results raise the possible use of this model in nanotoxicological studies of osseointegrated devices and demonstrate a possible therapeutic potential of this TiO2 NPs to prevent or reverse bone resorption.

Expression of metalloproteinases and their tissue inhibitors in inflamed gingival biopsies.

OBJECTIVES: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in the periodontal disease process. Results of in vivo MMPs and TIMPs gene expressions in the gingiva, though, are still controversial. In the present study, we compared the gene expression of MMP-1, -2, -9, -13 and TIMP-1, -2 in healthy and inflamed gingiva. METHODS: 38 gingival samples were collected from gingivitis (n = 10), advanced chronic periodontitis (n = 10), generalized aggressive periodontitis (n = 8) and periodontally healthy individuals (n = 10). Total RNA isolated from those samples was subjected to reverse transcription followed by amplification by polymerase chain reaction (RT-PCR). Products were visualized in agarose gels and quantified by optical densitometry. Samples were also processed for gelatin zymography and Western blotting for MMP-2 and MMP-9 in order to assess for post-transcriptional MMP regulation at the protein level. RESULTS: The frequencies and levels of transcripts encoding MMPs and TIMPs were found to be not significantly different among groups (p > 0.05, Fisher's Exact and Kruskall-Wallis tests). There is a trend towards higher MMP-2 and -9 gelatinase activities in the inflamed samples, although not statistically significant. In contrast, zymography and Western blotting studies show that MMP-2 is virtually absent in the chronic periodontitis group. CONCLUSION: These results could reflect a complex regulation of MMPs and TIMPs' gene expression in the course of gingival inflammation. They also reveal a great biological diversity even among individuals with similar periodontal status.

Defining subphenotypes for oral clefts based on dental development.

Individuals with clefts present considerably more dental anomalies than do individuals without clefts. We used dental development to subphenotype clefts with the goal of identifying cleft subgroups that could have specific genetic contributions. We examined 1000 individuals, 500 with clefts and 500 without. We used several clinical features, such as cleft completeness or incompleteness, laterality, and the presence of dental anomalies to assess each individual's cleft status. We performed chi-square and Fisher's exact tests to compare the frequencies of observed anomalies between individuals with and individuals without clefts, and among individuals with different cleft subphenotypes. Agenesis of the lateral incisor on the non-cleft side was the most remarkable observation, and may suggest that such cases could be considered incomplete forms of bilateral clefts of the lip.

Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

Bone morphogenetic proteins: from structure to clinical use.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ss superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications.

Titanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration.

Tooth agenesis association with self-reported family history of cancer.

It has been proposed that tooth agenesis and cancer development share common molecular pathways. We performed a cross-sectional study to investigate the epidemiological and molecular association between tooth agenesis and self-reported family history of cancer. Eighty-two individuals with tooth agenesis and 328 individuals with no birth defect were recruited from the same institution. Tooth agenesis was assessed in permanent teeth and was defined based on the age of the participants and when initial tooth formation should be radiographically visible. We also investigated the role of genes involved in dental development that have been implicated in tumorigenesis, and 14 markers in AXIN2, FGF3, FGF10, and FGFR2 were genotyped. Individuals with tooth agenesis had an increased risk of having a family history of cancer (p = 0.00006; OR = 2.7; 95% C.I., 1.6-4.4). There were associations between AXIN2, FGF3, FGF10, and FGFR2 with tooth agenesis [i.e., individuals who carried the polymorphic allele of FGFR2 (rs1219648) presented higher risk for having premolar agenesis (p = 0.02; OR = 1.8; 95% C.I., 1.1-3.0)]. In conclusion, tooth agenesis was associated with positive self-reported family history of cancer and with variants in AXIN2, FGF3, FGF10, and FGFR2. Prospective studies are needed to confirm if tooth agenesis can be used as a risk marker for cancer.

Candidate gene studies in hypodontia suggest role for FGF3.

INTRODUCTION: The majority of tooth agenesis cases are mild (hypodontia) and typically not associated with the gene mutations linked to oligodontia. From this, we hypothesise that most cases of tooth agenesis fit a polygenic mode of inheritance, where several genes with small effects cause a variety of varying phenotypes. MATERIALS AND METHODS: In this study, we looked at 18 not typically studied genes in this condition, to ascertain their contribution to hypodontia. Our study subjects consisted of 167 patients with hypodontia and their parents from two cohorts (one from Brazil and one from Turkey). An additional 465 DNA samples (93 cases with hypodontia and 372 controls without family history for tooth agenesis or oral clefts) from Brazil were also available for this study. Ninety-three single nucleotide polymorphisms that maximally represent the linkage disequilibrium structure of the genes for the 18 genes were selected and genotyped using Taqman chemistry. Chi square was used to test if genotype distributions were in Hardy-Weinberg equilibrium, and 24 markers that were in Hardy-Weinberg equilibrium and had allele frequencies higher than 5 % in a panel of 50 CEPH samples were further tested. Association between hypodontia and genetic variants was tested with the transmission disequilibrium test within the programme Family-Based Association Test (FBAT) and by using Chi square and Fisher's exact tests. Alpha at a level of 0.05 was used to report results. RESULTS: Results suggest possible associations between several genes and hypodontia in the three populations. In the Turkish cohort (n = 51 parent-affected child trios) the most significant results were as follows: FGF3 rs1893047, p = 0.08; GLI3 rs929387, p = 0.03; GLI3 haplotype rs929387-rs846266, p = 0.002; and PAX9 rs2073242, p = 0.03. In the Brazilian cohort (n = 116 parent-affected child trios), the results were as follows: DLX1 rs788173, p = 0.07; FGF3 rs12574452, p = 0.03; GLI2 rs1992901, p = 0.03; and PITX2 rs2595110, p = 0.01. The second Brazilian cohort also suggested that FGF3 (rs12574452, p = 0.01) is associated with hypodontia and added EDAR (rs17269487, p = 0.04), LHX6 (rs989798, p = 0.02), and MSX1 (rs12532, p = 0.003). CONCLUSION: Our results suggest that several genes are potentially associated with hypodontia and their individual contributions may be modest. Hence, these cases may not be explained by inactivating mutations such as many oligodontia cases segregating in a Mendelian fashion but rather are influenced by one or more susceptibility alleles in multiple small effect genes.

Novel cleft susceptibility genes in chromosome 6q.

Cleft lip/palate is a defect of craniofacial development. In previous reports, chromosome 6q has been suggested as a candidate region for cleft lip/palate. A multipoint posterior probability of linkage analysis of multiplex families from the Philippines attributed an 88% probability of harboring a cleft-susceptibility gene to a narrower region on bands 6q14.2-14.3. We genotyped 2732 individuals from families and unrelated individuals with and without clefts to investigate the existence of possible cleft-susceptibility genes in this region. We found association of PRSS35 and SNAP91 genes with cleft lip/palate in the case-control cohort and in Caucasian families. Haplotype analyses support the individual associations with PRSS35. We found Prss35 expression in the head and palate of mouse embryos at critical stages for palatogenesis, whereas Snap91 was expressed in the adult brain. We provide further evidence of the involvement of chromosome 6q in cleft lip/palate and suggest PRSS35 as a novel candidate gene.

Radiographic assessment of impacted mandibular third molar sockets filled with composite xenogenic bone graft.

OBJECTIVES: The main purpose of this study was to evaluate the radiographic aspect of the healing of extraction sockets filled with a xenogenic graft material (Gent-tech). METHODS: Thirty-nine patients ranging in age from 15 years to 25 years with bilateral impacted mandibular molars were chosen based on bilateral mandibular similarities. After tooth extraction, the socket was filled with the graft. The opposite site was left to heal naturally and served as a control. The experimental and control sites were chosen randomly. Bone density and crest healing were evaluated on digital radiographs taken immediately, 2 months and 6 months after surgery. The respective pixels values obtained with the Digora software were compared statistically. RESULTS: The results showed a significant decrease in the distance from the cemento-enamel junction to the alveolar bone crest, but no difference was found between the control and experimental groups. Bone density increased significantly, and there was difference between experimental and control groups. CONCLUSION: The analysed parameters observed by the authors were similar to those of the control group, suggesting xenogenic graft being an acceptable material and a graft option.

Analysis of fingernails and urine as biomarkers of fluoride exposure from dentifrice and varnish in 4- to 7-year-old children.

The use of fingernails and urine as biomarkers of exposure to fluoride (F) from fluoridated dentifrice and varnish was evaluated in twenty 4- to 7-year-old children, who were divided into two groups: group A (9 caries-free children) and group B (11 children with past caries experience). They used a placebo dentifrice for 28 days, fluoridated dentifrice (1,570 ppm F) for the following 28 days, and placebo dentifrice for an additional 28 days, then returned to their usual dentifrices. Group B children also received 4-week applications of a varnish (2.26% F) while using the fluoridated dentifrice. Urinary collections were performed 24 h before the use of fluoridated dentifrice and 24 h (group A) or 48 h (group B) after. Fingernails were clipped every 2 weeks, for 26 weeks. Total F intake from diet and dentifrice was estimated. Fingernail F concentrations did not vary significantly throughout the study. Twenty-four-hour urinary F outputs (mean +/- SD, microg) were: 414 +/- 200 and 468 +/- 253 for placebo and F dentifrices, respectively (group A) and 402 +/-206, 691 +/- 345, 492 +/- 243 for placebo dentifrice, F dentifrice plus F varnish and F dentifrice, respectively (group B). The use of F dentifrice did not cause a significant increase in the urinary F output. However, when F varnish was used, a transitory increase in the urinary F output was detected (p = 0.001), returning to baseline levels in the last 24 h. Thus, F varnish is a safe method for topical F application even in children that use F dentifrice regularly. According to our protocol, urine was a suitable biomarker of exposure to F from dentifrice plus varnish, but not from dentifrice alone, while nails were not.

Fluoride content of infant formulas prepared with deionized, bottled mineral and fluoridated drinking water.

Usually infant milk formula is the major source of fluoride in infancy. Fluoride concentrations in ten samples of powdered milk formulas, prepared with deionized, bottled mineral, and fluoridated drinking water were determined after HMDS-facilitated diffusion, using a fluoride ion specific electrode(Orion 9609). Fluoride concentrations ranged from 0.01 to 0.75 ppm; from 0.02 to 1.37 ppm and from 0.91 to 1.65 ppm for formulas prepared with deionized, bottled mineral (0.02 to 0.69 ppm F) and fluorinated drinking water (0.9 ppm F), respectively. Possible fluoride ingestion per Kg body mass ws estimated. With deionized water, only the soy-based- formulas should provide a daily fluoride intake of above the suggested threshold for fluorosis. With water containing 0.9 ppm F, however, all of them would provide it. Hence, to limit fluoride intakes to amounts <0.1 mg/kg/day, it is necessary to avoid use fo fluoridated water (around 1 ppm) to dilute powdered infant formulas.

Soybean seed acid phosphatases: unusual optimum temperature and thermal stability studies.

In contrast to other acid phosphatases, four cytoplasmic isoforms (AP1, AP2, AP3A, and AP3B) purified from mature soybean seeds presented high activities at temperatures above 80 degrees C, when p-nitrophenylphosphate (p-NPP) was utilized as substrate. However, with tyrosine phosphate and inorganic pyrophosphate as substrates, maximum activities were observed at temperature of 60 degrees C during 10 min reaction. In the absence of substrate, enzymes lost only 20% activity after 60 min at 60 degrees C; the isoforms AP3A and AP3B retained 30% of activity at 70 degrees C after 60 min and all the isoforms were inactivated at 80 degrees C, after 5 min. Thermal inactivation studies indicated that the soybean enzymes showed different temperature dependences in relation to most plant acid phosphatases. A best protective effect was observed when the isoforms were preincubated, at 70 degrees C, with phosphate (10 mM) and p-nitrophenol (10 mM) which indicates that the enzyme inactivation was prevented only in the presence of both reaction products.

Bone morphogenetic proteins: from structure to clinical use

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ß superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.