Doxycycline Decreases Atherosclerotic Lesions in the Aorta of ApoE-/- and Ovariectomized Mice with Correlation to Reduced MMP-2 Activity.

Atherogenic events promote changes in vessel walls, with alteration of the redox state, and increased activity of matrix metalloproteinases (MMPs). Thus, this study aims to evaluate aortic remodeling, MMP activity, and reactive oxygen species (ROS) levels after treatment with doxycycline in ApoE-/- and ovariectomized mice (OVX). Female ApoE-/--knockout mice (5 weeks) were submitted to ovariectomy surgery to induce experimental menopause. They then received chow enriched with 1% cholesterol to induce hypercholesterolemia. The animals were divided into two experimental groups: ApoE-/-/OVX vehicle and ApoE-/-/OVX doxycycline (30 mg/kg) administered by gavage once a day for 28 days (15th to the 18th week of life). Blood samples were collected to measure total cholesterol and fractions. The aorta was used for morphometry and to measure the activity and expression of MMP-2 and ROS levels. The ApoE-/-/OVX doxycycline group showed no change in total and fraction cholesterol levels. However, there was a reduction in ROS levels, MMP-2 expression, and activity that correlated with a decrease in atherosclerotic lesions relative to the ApoE-/-/OVX vehicle (p > 0.05). Therefore, we conclude that doxycycline in ApoE-/-/OVX animals promotes a reduction in atherosclerotic lesions by reducing ROS and MMP-2 activity and expression.

Naringenin mitigates titanium dioxide (TiO2)-induced chronic arthritis in mice: role of oxidative stress, cytokines, and NFκB.

OBJECTIVE: To evaluate the effect and mechanisms of naringenin in TiO2-induced chronic arthritis in mice, a model resembling prosthesis and implant inflammation. TREATMENT: Flavonoids are antioxidant and anti-inflammatory molecules with important anti-inflammatory effect. Mice were daily treated with the flavonoid naringenin (16.7-150 mg/kg, orally) for 30 days starting 24 h after intra-articular knee injection of 3 mg of TiO2. METHODS: TiO2-induced arthritis resembles cases of aseptic inflammation induced by prosthesis and/or implants. Mice were stimulated with 3 mg of TiO2 and after 24 h mice started to be treated with naringenin. The disease phenotype, treatment toxicity, histopathological damage, oxidative stress, cytokine expression and NFκB were evaluated after 30 days of treatment. RESULTS: Naringenin inhibited TiO2-induced mechanical hyperalgesia (96%), edema (77%) and leukocyte recruitment (74%) without inducing toxicity. Naringenin inhibited histopathological index (HE, 49%), cartilage damage (Toluidine blue tibial staining 49%, and proteoglycan 98%), and bone resorption (TRAP-stained 73%). These effects were accompanied by inhibition of oxidative stress (gp91phox 93%, NBT 83%, and TBARS 41%) cytokine mRNA expression (IL-33 82%, TNFα 76%, pro-IL-1ß 100%, and IL-6 61%), and NFκB activation (100%). CONCLUSION: Naringenin ameliorates TiO2-induced chronic arthritis inducing analgesic and anti-inflammatory responses with improvement in the histopathological index, cartilage damage, and bone resorption.

The flavonoid quercetin inhibits titanium dioxide (TiO2)-induced chronic arthritis in mice.

Titanium dioxide (TiO2) is a common component of orthopedic prosthesis. However, prosthesis wear releases TiO2, which induces inflammation and osteolysis in peri-prosthetic tissues. Quercetin is a flavonoid widely present in human diet, which presents biological activities such as antinociceptive, anti-inflammatory and antioxidant effects. Therefore, the effect of intraperitoneal treatment with quercetin in TiO2-induced arthritis model was evaluated. In the first set of experiments, mice received injection of TiO2 (0.1-3 mg/knee joint) and articular mechanical hyperalgesia, edema and histopathology analysis were performed in a 30 days protocol. The dose of 3 mg of TiO2 showed the most harmful effect, and was chosen to the following experiments. Subsequently, mice received 3 mg of TiO2 followed by post-treatment with quercetin during 30 days. Quercetin (10-100 mg/kg) inhibited in a dose-dependent manner TiO2-induced knee joint mechanical hyperalgesia, edema and leukocyte recruitment and did not induce damage in major organs such as liver, kidney and stomach. The dose of 30 mg/kg was chosen for the subsequent analysis, and reduced histopathological changes such as leukocyte infiltration, vascular proliferation and synovial hyperplasia (pannus formation) on day 30 after TiO2 challenge. The protective analgesic and anti-inflammatory mechanisms of quercetin included the inhibition of TiO2-induced neutrophil and macrophage recruitment, proteoglycan degradation, oxidative stress, cytokine production (TNF-α, IL-1ß, IL-6, and IL-10), COX-2 mRNA expression, and bone resorption as well as activation of Nrf2/HO-1 signaling pathway. These results demonstrate the potential therapeutic applicability of the dietary flavonoid quercetin to reduce pain and inflammatory damages associated with prosthesis wear process-induced arthritis.

* Bone Regeneration Mediated by a Bioactive and Biodegradable Extracellular Matrix-Like Hydrogel Based on Elastin-Like Recombinamers.

The morbidity of bone fractures and defects is steadily increasing due to changes in the age pyramid. As such, novel biomaterials that are able to promote the healing and regeneration of injured bones are needed to overcome the limitations of auto-, allo-, and xenografts, while providing a ready-to-use product that may help to minimize surgical invasiveness and duration. In this regard, recombinant biomaterials, such as elastin-like recombinamers (ELRs), are very promising as their design can be tailored by genetic engineering, thus allowing scalable production and batch-to-batch consistency, among others. Furthermore, they can self-assemble into physically crosslinked hydrogels above a certain transition temperature, in this case body temperature, but are injectable below this temperature, thereby markedly reducing surgical invasiveness. In this study, we have developed two bioactive hydrogel-forming ELRs, one including the osteogenic and osteoinductive bone morphogenetic protein-2 (BMP-2) and the other the Arg-Gly-Asp (RGD) cell adhesion motif. The combination of these two novel ELRs results in a BMP-2-loaded extracellular matrix-like hydrogel. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand white rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neoformation was confirmed using different techniques, such as radiography, computed tomography, and histology. This hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone.

Lead line in rodents: an old sign of lead intoxication turned into a new method for environmental surveillance.

The "lead line" was described by Henry Burton in 1840. Rodents are used as sentinels to monitor environmental pollution, but their teeth have not been used to determine lead. To determine whether lead deposits can be observed in the teeth of lead-exposed animals, since the gingival deposits known as "lead line" would likely have a correlate in the calcified tissue to which the gums are opposed during life. Male Wistar rats were exposed to lead in the drinking water (30 mg/L) since birth until 60 days-old. Molars and the incisors of each hemimandible were analyzed by scanning electron microscopy (SEM) on regular and backscattered electrons (BSE) mode. Elements were determined using electron dispersive spectroscopy (EDS). Clean cervical margins were observed on control teeth, as opposed to the findings of extensive deposits on lead-exposed animals, even in hemimandibles that had been exhumed after being buried for 90 days. BSE/EDS indicated that those deposits were an exogenous material compatible with lead sulfite. Presence of calcium, phosphorus, magnesium, carbon, lead, and oxygen is presented. Lead-exposed animals presented marked root resorption. The lead deposits characterized here for the first time show that the "lead line" seen in gums has a calcified tissue counterpart, that is detectable post-mortem even in animals exposed to a low dose of lead. This is likely a good method to detect undue lead exposure and will likely have wide application for pollution surveillance using sentinels.

Bone Recuperation After rhBMP-2 Insertion in Alcoholic Animals-Experimental Study.

BACKGROUND: Alcoholism affects bone repair and this study evaluated the recombinant human BMP-2 (rhBMP-2)/collagen sponge association aiming to improve the bone healing process. The aim of this study was to investigate the action of alcoholism and its effect on the repair of bone defects (BD) performed on rat calvaria after the application of rhBMP-2, either pure or combined with a collagen matrix, using radiographic, histological and immunohistochemical methods. METHODS: We used 80 rats divided into two groups and these into 4 subgroups, each with a waiting period for sacrifice of four and six weeks after the BD (5mm). The groups were divided into: Veh-X) vehicle+BD, Veh-BMP) water+BD+5µg rhBMP-2, Veh-ACS) water+ BD+absorbable collagen sponge, Veh-BMP/ACS) water+BD+5µg rhBMP-2/absorbable collagen sponge, EtOH-X) ethanol+BD, EtOH-BMP) ethanol+BD+5µgrhBMP-2, EtOH-ACS) ethanol+BD+absorbable collagen sponge, and EtOH-BMP/ACS) ethanol+ BD+5µg of rhBMP-2/ absorbable collagen sponge. RESULTS: Radiographically, it was found that after six weeks, for the groups treated with rhBMP-2, independent of the carrier use and ethanol administration, there was more new bone formation (p<0.05). For immunohistochemical analysis, osteocalcin and bone sialoprotein were found to be predominant in groups treated with rhBMP-2. For quantitative stereology, which aims to calculate the volume of new bone, higher values for the groups treated with rhBMP-2 pure or combined with the carrier were found; but for the groups treated with ethanol, a higher bone formation in the groups treated with rhBMP-2 associated with the carrier in the periods of four and six weeks (p<0.001) was found. CONCLUSION: It was concluded that the carrier was effective for rhBMP-2 delivery, even in the presence of ethanol.

Matrix metalloproteinases are involved in cardiovascular diseases.

This MiniReview describes the essential biochemical and molecular aspects of matrix metalloproteinases (MMPs) and briefly discusses how they engage in different diseases, with particular emphasis on cardiovascular diseases. There is compelling scientific evidence that many MMPs, especially MMP-2, play important roles in the development of cardiovascular diseases; inhibition of these enzymes is beneficial to many cardiovascular conditions, sometimes precluding or postponing end-organ damage and fatal outcomes. Conducting comprehensive discussions and further studies on how MMPs participate in cardiovascular diseases is important, because inhibition of these enzymes may be an alternative or an adjuvant for current cardiovascular disease therapy.

Biological evaluation of the bone healing process after application of two potentially osteogenic proteins: an animal experimental model.

OBJECTIVE: The aim of this work was to analyse qualitatively and quantitatively the newly formed bone after insertion of rhBMP-2 and protein extracted from Hevea brasiliensis (P-1), associated or not with a carrier in critical bone defects created in Wistar rat calvarial bone, using histological and histomorphometrical analyses. MATERIALS AND METHODS: Eighty-four male Wistar rats were used, divided into two groups, according to the period of time until the sacrifice (2 and 6 weeks). Each one of these groups was subdivided into six groups with seven animals each, according to the treatments: (1) 5 µg of pure rhBMP-2, (2) 5 µg of rhBMP-2/monoolein gel, (3) pure monoolein gel, (4) 5 µg of pure P-1, (5) 5 µg of P-1/monoolein gel and (6) critical bone defect controls. The animals were euthanised and the calvarial bone tissue removed for histological and histomorphometrical analyses. RESULT AND CONCLUSION: The results showed an improvement in the bone healing process using the rhBMP-2 protein, associated or not with a material carrier in relation to the other groups, and this process demonstrated to be time dependent.

Influence of decortication of the recipient graft bed on graft integration and tissue neoformation in the graft-recipient bed interface.

The objective of the present study was to assess the influence of decortication of the posterior elements of the vertebra (recipient bed) and the nature of the bone graft (cortical or cancellous bone) on graft integration and bone, cartilage and fiber neoformation in the interface between the vertebral recipient bed and the bone graft. Seventy-two male Wistar rats were divided into four experimental groups according to the presence or absence of decortication of the posterior vertebral elements and the use of a cortical or cancellous bone graft. Group I--the posterior elements were decorticated and cancellous bone used. Group II--the posterior elements were decorticated and cortical graft was used. Group III--the posterior elements were not decorticated and cancellous graft was used. Group IV--the posterior elements were not decorticated and cortical graft was used. The animals were killed 3, 6 and 9 weeks after surgery and the interface between the posterior elements and the bone graft was subjected to histomorphometric evaluation. Mean percent neoformed bone was 40.8% in group I (decortication and cancellous graft), 39.13% in group II (decortication and cortical graft), 6.13% in group III (non-decorticated and cancellous graft), and 9.27% in group IV (non-decorticated and cortical graft) for animals killed at 3 weeks (P = 0.0005). For animals killed at 6 weeks, the mean percent was 38.53% for group I, 40.40% for group II, 10.27% for group III, and 7.6% for group IV (P = 0.0005), and for animals killed at 9 weeks, the mean was 25.93% for group I, 30.6% for group II, 16.4% for group III, and 18.73% for group IV (P = 0.0026). The mean percent neoformed cartilage tissue was 8.36% for group I, 7.46% for group II, 11.1% for group III, and 9.13% for group IV for the animals killed at 3 weeks (P = 0.6544); 6.6% for group I, 8.07% for group, 7.47% for group III and 6.13% for group IV (P = 0.4889) for animals killed at 6 weeks, and 3.13% for group I, 4.06% for group II, 10.53% for group III and 12.07% for group IV (P = 0.0006) for animals killed at 9 weeks. Mean percent neoformed fibrous tissue was 11% for group I, 6.13% for group II, 26.27% for group III and 21.87% for group IV for animals killed at 3 weeks (P = 0.0008); 7.67% for group I, 7.1% for group II, 9.8% for group III and 10.4% for group IV (P = 0.7880) for animals killed at 6 weeks, and 3.73% for group I, 4.4% for group II, 6.67% for group III and 6.8% for group IV (P = 0.0214) for animals killed at 9 weeks. The statistically significant differences in percent tissue formation were related to decortication of the posterior elements. The use of a cortical or cancellous graft did not influence tissue neoformation. Ossification in the interface of the recipient graft bed was of the intramembranous type in the decorticated animals and endochondral type in the non-decorticated animals.