Rodrigo Cardoso de Oliveira

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.

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss‐of‐function mutations within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl−/−, aka Akp2−/−) mice closely phenocopy infantile HPP, including the rickets, vitamin B6‐responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl−/− mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral‐targeted TNAP (ENB‐0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage‐specific expression of TNAP in ameloblasts. In the Alpl−/− mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter‐rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral‐targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral‐targeted TNAP to prevent enamel defects in HPP.

Effect of Iron on Matrix Metalloproteinase Inhibition and on the Prevention of Dentine Erosion

It is known that some metal salts can inhibit matrix metalloproteinase (MMP) activity, but the effect of iron has not been tested yet. On the other hand, it has recently been suggested that MMP inhibition might influence dentine erosion. Based on this, the aims of this study were: (1) to test in vitro the effect of FeSO4 on MMP-2 and -9 activity, and (2) to evaluate in situ the effect of FeSO4 gel on dentine erosion. MMP-2 and -9 activities were analysed zymographically in buffers containing FeSO4 in concentrations ranging between 0.05 and 1.5 mmol/l or not. Volunteers (n = 10) wore devices containing bovine dentine blocks (n = 60) previously treated with the following gel treatments: FeSO4 (1 mmol/l FeSO4), F (NaF 1.23%; positive control) and placebo (negative control). The gels were applied once and removed after 1 min. Erosion was performed extraorally with Coca-Cola 4 times per day for 5 min over 5 days. Dentine wear was evaluated by profilometry. The data were analysed by Kruskal-Wallis and Dunn’s tests (p < 0.05). FeSO4 inhibited both MMP-2 (IC50 = 0.75 mmol/l) and MMP-9 (IC50 = 0.50 mmol/l) activities. In the in situexperiment, the mean wear (± SD) found for the F gel (0.79 ± 0.08 µm) was significantly reduced in more than 50% when compared to the placebo gel (1.77 ± 0.33 µm), but the FeSO4 gel completely inhibited the wear (0.05 ± 0.02 µm). Since FeSO4 was able to inhibit MMP in vitro, it is possible that the prevention of dentine wear by the FeSO4 gel in situ might be due to MMP inhibition, which should be investigated in further studies.

Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode.

Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm(2)), infrared laser (780 nm, 40 mW, 1 W/cm(2)), and red LED (637 ± 15 nm, 40 mW, 1 W/cm(2)). All applications were punctual and performed with a spot with 0.4 mm(2) of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p ≤ 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced.

Proteomic analysis of kidney in rats chronically exposed to fluoride.

Two-dimensional gel electrophoresis (2-DE) was used to better understand alterations in renal metabolism induced by fluoride (F). Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F for 60 days (n=6/group). Kidneys were collected for proteomic and histological (HE) analysis. After protein isolation, renal proteome profiles were examined using 2-DE and Colloidal Coomassie Blue staining. Protein spots with a 2-fold significant difference as detected by quantitative intensity analysis (Image Master Platinum software) and t-test (p<0.05) were excised and analyzed by MALDI-TOF MS (matrix assisted laser desorption ionization-time-of-flight mass spectrometry). The histological analysis revealed no damage in kidneys induced by F, except for a vascular congestion in the 50 ppm F group. Between control vs 50 ppm F, and control vs 5 ppm F groups, 12 and 6 differentially expressed proteins were detected, respectively. Six proteins, mainly related with metabolism, detoxification and housekeeping, were successfully identified. At the high F group, pyruvate carboxylase, a protein involved in the formation of oxaloacetate was found to be downregulated, while enoyl coenzyme A hydratase, involved in fatty acids oxidation, was found to be upregulated. Thus, proteomic analysis can provide new insights into the alterations in renal metabolism after F exposure, even in low doses.

Tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats

The treatment of persistent bone defects has encouraged the search for proper techniques or bone substitutes. In Dentistry, a common problem in the treatment of periodontal bone defects is the growth of tissues within the lesion, such as the junctional epithelium, which impair regeneration of these tissues. Guided tissue regeneration (GTR), based on the separation of the tissues by means of membranes or barriers, was developed in an attempt to improve periodontal regeneration. The aim of this study was to histologically evaluate the tissue response to a membrane of demineralized bovine cortical bone implanted in the subcutaneous tissue of rats. The study periods were 1, 3, 7, 15, 30 and 60 days after implantation. Analysis of the histological sections demonstrated a moderate to intense inflammatory response at 1 and 3 days, moderate at 7 and 15 days, and almost absent at 30 and 60 days. Resorption of the membrane began 15 days after implantation, and at 60 days only remnants could be detected in some animals. We concluded that the demineralized bovine cortical bone membrane was well tolerated by the tissues and is completely resorbed after 30-60 days by mononuclear cells and multinucleated giant cells, which disappear upon completion of the process.

The effect of different fluoride concentrations and pH of dentifrices on plaque and nail fluoride levels in young children.

To evaluate the influence of dentifrice pH and fluoride (F) concentration on F uptake by plaque and nails, two sets of 5- to 6-year-old children were randomly allocated into four groups, according to the type of dentifrice they had been using for 1 year: (1) experimental liquid dentifrice (ELD), 1,100 ppm F, pH 7.0; (2) ELD, 1,100 ppm F, pH 4.5; (3) ELD, 550 ppm F, pH 4.5, and (4) commercial toothpaste, 1,100 ppm F, pH 7.0. In one set of children, nails were clipped. In the second, plaque samples were collected 1 h after the last use of dentifrice. F concentration in plaque and nails was analyzed. Plaque F concentration was significantly lower in group 4 than in groups 1–3. Nail F concentration was significantly higher in group 4, and significantly lower in group 3, than in group 1 or 2. Plaque F uptake was influenced significantly by dentifrice consistency and nonsignificantly by pH and F concentration. Reduction of dentifrice pH did not affect nail F concentration.

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

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

Microscopic and radiographic analysis of the effect of particle size of demineralized bovine cancellous bone matrix on the repair of bone defects in femurs of rabbits

The bone tissue has a great regenerative potential, with ability to completely restore its structure and original functions. In some situations, though, bone defects cannot be self-repaired, thus requiring the use of grafts for a correct treatment and good prognosis. This work aimed at microscopically analyzing the effect of the particle size of demineralized bovine cancellous bone matrix in micro and macrogranular forms on the repair of bone defects in femurs of rabbits, with blood clot used as control. At 1, 3 and 6 months after implantation of the materials, the animals were killed and the anatomic specimens were removed. A foreign body-type granulomatous reaction containing macrophages and multinucleated giant cells in contact with the implanted particles was observed. These results suggest a failure in demineralization and/or interruption of the antigenic potential during production of the biomaterial. It is concluded that the size of the particles did not influence the evolution of the repair process of bone defects, acting only as bone-filler substances, and that the material implanted should be improved by quality control during production, since it may represent a good alternative for bone graft.

Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers.