Healing outcomes at implants installed in sites augmented with particulate autologous bone and xenografts. An experimental study in dogs

Aim: To evaluate the integration of implants installed at the interface of pristine and grafted tissue augmented with particulate autologous bone or deproteinized bovine bone mineral (DBBM), concomitantly with a collagen membrane. Material and methods: In 6 Labrador dogs, the distal root of 3P3 and 4P4 was endodontically treated and hemi-sected, and the mesial roots extracted concomitantly with the extraction of 2P2. The buccal bony walls were removed, and two box-shaped defects, one larger and one smaller, were created. After 3 months, flaps were elevated, and the defects were filled with particulate autologous bone or DBBM in the right and left side of the mandible, respectively. Collagen membranes were used to cover the grafted areas. Three months later, flaps were elevated, and a customized device was used as surgical guide to prepare the recipient sites at the interface between grafts and pristine bone. One implant was installed in each of the four defects. After 3 months, biopsies were harvested and ground sections prepared for histological evaluation. Results: The augmentation technique was effective at all sites and all the foreseen implants were installed. In the histological analysis, all implants were integrated in mature bone, at both the buccal and lingual aspects. The most coronal bone-to-implant contact and the top of the buccal bony crest were located at a similar distance between test and control implants. However, these distances were higher at the larger compared with the smaller defects. Especially in the large defect, residual particles of DBBM were found embedded into connective tissue and located outside the bony crest. Conclusions: Particulate autologous bone as well as DBBM particles used to augment horizontally the alveolar bony process allowed for the osseointegration of implants installed after 3 months of healing. © 2012 John Wiley & Sons A/S.

Kinematic analysis in healthy and hip-dysplastic German Shepherd dogs

This study investigated kinematic patterns in clinically normal German Shepherd dogs (GSDs) compared to those with hip dysplasia and with no clinical signs of lameness. Two groups of GSDs, including 10 clinically healthy dogs (G1) and 10 with hip dysplasia (G2), were trotted on a treadmill at a constant speed. Kinematic data were collected by a 3-camera system and analysed by a motion-analysis program. Flexion and extension joint angles and angular velocities were determined for the shoulder, elbow, carpal, hip, stifle, and tarsal joints.Within each group, the differences between the right and left limbs in all kinematic variables were not significant. Minimum angle, angular displacement and minimum angular velocity did not differ between groups. Significant differences were observed in the maximum angular velocity and maximum angle of the hip joint (dysplastic. >. healthy), and in the maximum angular velocity of the carpal joint (healthy. >. dysplastic). It was concluded that, when trotting on a treadmill, dysplastic dogs with no signs of lameness may present joint kinematic alterations in the hind as well as the forelimbs. © 2012 Elsevier Ltd.

Comparisons between Bio-Oss® and Straumann® Bone Ceramic in immediate and staged implant placement in dogs mandible bone defects

Objective: To compare immediate and staged approach implant placement in circumferential defects treated with deproteinized bovine bone mineral (DBBM); hidroxyapatite/tricalcium phosphate (HA/TP); autogenous bone (Ab); and coagulum (Cg); upon implant stability, osseointegration and alveolar crest maintenance. Materials and methods: Six dogs underwent extractions of lower premolars, bilaterally. Twelve weeks later four bone defects (6 mm wide/4 mm long) were drilled at one side and randomly filled with DBBM; HA/TP; Ab; and Cg, respectively, and left to heal (staged approach). Eight weeks later one implant (Osseospeed™, AstraTech) was placed in experimental sites. At the same session four defects were drilled on contra-lateral side and implants were inserted immediately after biomaterials grafting (immediate approach). Animals were euthanized 8 weeks later. Implant stability was measured by resonance frequency analysis (RFA) at installation and after sacrifice. Ground sections were prepared for bone contact (BIC); bone area (BA); distance implant shoulder-bone crest (IS-C); distance implant shoulder first bone contact (IS-B); and areas occupied by soft tissue. Results: The BA and BIC were superior in the staged approach. The Cg exhibited higher BIC and BA as compared with other materials at the total implant body (P = 0.004 and 0.012, respectively). The DBBM, HA/TP and Ab groups rendered similar BA and BIC. The immediate approach resulted in less crest resorption compared to staged approach. The biomaterials did not affect the IS-C and IS-B measurements. Particles area tended to be higher in DBBM group than HA/TP (P = 0.15), while soft tissue infiltrate was higher in DBBM group when used in the immediate approach (P = 0.04). The RFA indicated gain in stability in the staged approach (P = 0.002). The correlation test between RFA vs. BIC and BA demonstrated inferior stability for DBBM group in immediate approach (P = 0.01). Conclusions: Implants placed in healed defects resulted in better stability as a consequence of higher BIC and BA. The Cg alone rendered increased BIC compared to other materials in both approaches. Immediate approach should be preferable to staged approach in terms of alveolar crest maintenance. The BIC and BA values did not vary between micro and macro-threads in this experimental model. Implants installed in sites filled with DBBM in immediate approach were less stable. © 2011 John Wiley & Sons A/S.

Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin. © 2012 Elsevier B.V.

Pro-inflammatory cytokines predominate in the brains of dogs with visceral leishmaniasis: A natural model of neuroinflammation during systemic parasitic infection

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. To investigate the pathogenesis of brain alterations occurring during visceral leishmaniasis infection, the expression of the cytokines IL-1β, IL-6, IL-10, IL-12p40, IFN-γ, TGF-β and TNF-α and their correlations with peripheral parasite load were evaluated in the brains of dogs naturally infected with Leishmania infantum. IL-1β, IFN-γ and TNF-α were noticeably up-regulated, and IL-10, TGF-β and IL-12p40 were down-regulated in the brains of infected dogs. Expression levels did not correlate with parasite load suggestive that the brain alterations are due to the host's immune response regardless of the phase of the disease. These data indicate the presence of a pro-inflammatory status in the nervous milieu of dogs with visceral leishmaniasis especially because IL-1β and TNF-α are considered key factors for the initiation, maintenance and persistence of inflammation. © 2012 Elsevier B.V.

Healing of buccal dehiscence defects at implants installed immediately into extraction sockets - an experimental study in dogs

Aim: To evaluate the influence of implant positioning into extraction sockets on bone formation at buccal alveolar dehiscence defects. Material and Methods: In six Labrador dogs the pulp tissue of the mesial roots of 4P4 was removed and the root canals were filled. Flaps were elevated bilaterally, the premolars hemi-sectioned and the distal roots removed. The implants were placed in contact with either the buccal (test site) or with the lingual (control site) bony wall of the extraction sockets. Healing abutments were affixed and triangular buccal bony dehiscence defects, about 2.7 mm deep and 3.5 mm wide, were then prepared. No regenerative procedures were done and a non-submerged healing was allowed. After 4 months of healing, block sections of the implant sites were obtained for histological processing and peri-implant tissue assessment. Results: After 4 months of healing, the bony crest and the coronal border of osseointegration at the test sites were located 1.71 ± 1.20 and 2.50 ± 1.21 mm apically to the implant shoulder, respectively. At the control sites, the corresponding values were 0.68 ± 0.63 and 1.69 ± 0.99 mm, respectively. The differences between test and control reached statistical significance (P < 0.05). Residual marginal bone defects were found both at the test and control sites. A statistically significant difference between test and control sites was only found at the lingual aspects (depth 2.09 ± 1.01 and 1.01 ± 0.48 mm, respectively). Similar heights of the buccal biological width were observed at both sites (about 5.1 mm). Conclusions: The placement of implants in a lingual position of the extraction sockets allowed a higher degree of bone formation at buccal alveolar dehiscence defects compared with a buccal positioning. © 2012 John Wiley & Sons A/S.

Influence of buccal bony crest width on marginal dimensions of peri-implant hard and soft tissues after implant installation. An experimental study in dogs

Aim: To evaluate the influence of the width of the buccal bony wall on hard and soft tissue dimensions following implant installation. Material and methods: Mandibular premolars and first molars of six Labrador dogs were extracted bilaterally. After 3 months of healing, two recipient sites, one on each side of the mandible, were prepared in such a way as to obtain a buccal bony ridge width of about 2 mm in the right (control) and 1 mm in the left sides (test), respectively. Implants were installed with the coronal margin flush with the buccal alveolar bony crest. Abutments were placed and the flaps were sutured to allow a non-submerged healing. After 3 months, the animals were euthanized and ground sections obtained. Results: All implants were completely osseointegrated. In respect to the coronal rough margin of the implant, the most coronal bone-to-implant contact was apically located 1.04 ± 0.91 and 0.94 ± 0.87 mm at the test and control sites, respectively, whereas the top of the bony crest was located 0.30 ± 0.40 mm at the test and 0.57 ± 0.49 mm at the control sites. No statistically significant differences were found. A larger horizontal bone resorption, however, evaluated 1 mm apically to the rough margin, was found at the control (1.1 ± 0.7 mm) compared to the test (0.3 ± 0.3 mm) sites, the difference being statistically significant. A thin peri-implant mucosa (2.4-2.6 mm) was found at implant installation while, after 3 months of healing, a biological width of 3.90-4.40 mm was observed with no statistically significant differences between control and test sites. Conclusions: A width of the buccal bony wall of 1or 2 mm at implant sites yielded similar results after 3 months of healing in relation of hard tissue and soft tissues dimensions after implant installation. © 2012 John Wiley & Sons A/S.

Increase nitric oxide and oxidative stress in dogs experimentally infected by Ehrlichia canis: Effect on the pathogenesis of the disease

The aim of this study was to evaluate nitric oxide levels, lipid peroxidation, protein oxidation and glutathione reductase activity in serum of dogs experimentally infected by Ehrlichia canis. Banked serum samples of dogs divided into two groups were used: negative control (n=5) and infected by E. canis (n=5). The concentration of nitrite/nitrate (NOx), lipid peroxidation (TBARS), advanced oxidation protein products (AOPP), and glutathione reductase (GR) activity in sera were evaluated. Samples were collected on days 0, 3, 6, 18 and 30 post-infection (PI). NOx and TBARS levels were significantly (P<0.05) higher in the infected group at 18 and 30 days PI, as well as AOPP levels at 30 days PI when compared to samples from control group. The GR activity was significant (P<0.05) increased in serum of dogs infected by E. canis on days 18 and 30 PI. Based on the increased levels of NOx, TBARS, AOPP and GR activity we concluded that dogs experimentally infected by E. canis develop a state of redox imbalance and that these changes might be involved in the pathophysiology of the disease. © 2013 Elsevier B.V.

Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease

Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production.

Deproteinized bovine bone mineral particles and osseointegration of implants without primary bone contact: An experimental study in dogs

Objectives: To evaluate the influence on osseointegration of Deproteinized bovine bone mineral (DBBM) particles used to fill defects of at least 1 mm around implants having no primary contact with bone. Material and methods: Premolars and first molars were extracted bilaterally from the mandible of six Labrador dogs. After 3 months of healing, mucoperiosteal full-thickness flaps were elevated, and one recipient site was prepared in the molar region of each hemi-mandible to place implants. These were installed with a deliberate circumferential and periapical space to the bone walls of 1.2 mm. All implants were stabilized with passive fixation plates to maintain the implants in situ and without any contact with the implant bed. The control sites were left to be filled with coagulum, while at the test sites, the residual gap was filled with DBBM. After 3 months of submerged healing, the animals were sacrificed. Ground sections were prepared and analyzed histomorphometrically. Results: Mineralized bone-to-implant contact was 4.0% and 3.9% for control and test sites, respectively. The width of the residual defects was 0.48 mm and 0.88 mm at the control and test sites, respectively. The percentage of implant surface covered by a layer of dense connective tissue of 0.12 mm of width on average was 84.9% and 88.5% at the control and test sites, respectively. Conclusion: A minor and not predictable degree of contact or distance osteogenesis was obtained on the implant surface when primary contact of the implant surface with the implant bed had deliberately been avoided. DBBM grafting of the artificial gap did not favor osseointegration. Neither did it enhance the ability to bridge the gap with newly formed bone in an artificial defect wider than 1 mm. © 2013 John Wiley & Sons A/S.

Enzyme effects on extruded diets for dogs with soybean meal as a substitute for poultry by-product meal

The effects of exogenous enzymes supplementation on kibble diets for dogs formulated with soybean meal (SBM) as a substitute for poultry by-product meal (PM) was investigated on nutrient digestibility, fermentation products formation, post-prandial urea response and selected faecal bacteria counts. Two kibble diets with similar compositions were used in two trials: PM-based diet (28.9% of PM; soybean hulls as a fibre source) and SBM-based diet (29.9% of SBM). In experiment 1, the SBM diet was divided into three diets: SBM-0, without enzyme addition; SBM-1, covered after extrusion with 7500U protease/kg and 45U cellulase/kg; and SBM-2, covered with 15000U protease/kg and 90U cellulase/kg. In experiment 2, the SBM diet was divided into three diets: SBM-0; SBM-1, covered with 140U protease/kg; 8U cellulase/kg, 800U pectinase/kg, 60U phytase/kg, 40U betaglucanase/kg and 20U xylanase/kg; and SMB-2, covered with 700U protease/kg, 40U cellulase/kg, 4000U pectinase/kg, 300U phytase/kg, 200U betaglucanase/kg and 100U xylanase/kg. Each experiment followed a block design with six dogs per diet. Data were submitted to analysis of variance and means compared by orthogonal and polynomial contrasts (p<0.05). In both experiments, nutrients and energy digestibility did not differ between diets (p>0.05). SBM consumption resulted in increased faecal moisture and production (p<0.05), without effect on faecal score. Higher concentration of propionate, acetate and lactate, and lower ammonia and pH were found in the faeces of dogs fed SBM (p<0.05). Higher post-prandial urea was verified in dogs fed SBM (p<0.05). In experiment 2, the addition of enzymes increased faecal concentration of propionate, acetate and total short-chain fatty acid (p<0.05) and tended to reduce post-prandial urea concentration (p=0.06). Although with similar digestibility, SBM shows a worse utilization of absorbed amino acids than the PM. Soybean oligosaccharides can beneficially change gut fermentation product formation. Enzymes can increase the gut fermentation activity and improve the SBM proteic value. © 2013 Blackwell Verlag GmbH.

Enzyme use in kibble diets formulated with wheat bran for dogs: Effects on processing and digestibility

Recently, there is an interest in technologies that favour the use of coproducts for animal nutrition. The effect of adding two enzyme mixtures in diets for dogs formulated with wheat bran (WB) was evaluated. Two foods with similar compositions were formulated: negative control (NC; without WB) and test diet (25% of WB). The test diet was divided into four treatments: without enzyme (positive control), enzyme mixture 1 (ENZ1; added before extrusion β-glucanase, xylanase, cellulase, glucoamylase, phytase); enzyme mixture 2 (ENZ2; added before extrusion the ENZ1 more α-amylase); enzyme mixture 2 added after the extrusion (ENZ2ex). ENZ1 and ENZ2 were used to evaluate the enzyme effect on extruder pre-conditioner (processing additive) and ENZ2ex to evaluate the effect of enzyme supplementation for the animal. Digestibility was measured through total collection of faeces and urine. The experiment followed a randomized block design with five treatments (diets) and six dogs per diet, totalling 30 dogs (7.0 ± 1.2 years old and 11.0 ± 2.2 kg of body weight). Data were submitted to analysis of variance and means compared by Tukey's test and orthogonal contrasts (p < 0.05). Reducing sugars showed an important reduction after extrusion, suggesting the formation of carbohydrate complexes. The apparent total tract digestibility (ATTD) of dry matter, organic matter, crude protein, acid-hydrolysed fat and energy was higher in NC than in diets with WB (p < 0.001), without effects of enzyme additions. WB diets resulted in higher faecal production and concentration of short-chain fatty acids (SCFA) and reduced pH and ammonia concentration (p < 0.01), with no effect of enzyme addition. The enzyme addition did not result in improved digestibility of a diet high in non-starch polysaccharides; however, only ATTD was measured and nutrient fermentation in the large intestine may have interfered with the results obtained. WB modified fermentation product formation in the colon of dogs. © 2013 Blackwell Verlag GmbH.

T and B lymphocytes in the brains of dogs with concomitant seropositivity to three pathogenic protozoans: Leishmania chagasi, Toxoplasma gondii and Neospora caninum

Background: Visceral leishmaniasis is a disease with great variability regarding the clinical manifestations in humans and dogs. Chronically infected dogs may develop neurological disorders, however, there are few reports that characterize the lesions and make clear the pathogenesis of the canine cerebral leishmaniasis. Concomitant with Leishmania chagasi, dogs may be infected by opportunistic pathogens, such as Toxoplasma gondii and Neospora caninum, which may contribute to the occurrence of lesions in the central nervous system. Hence, we aimed to compare the T and B lymphocytes population in the brains of infected dogs with seropositivity to L. chagasi, T. gondii and N. caninum concurrently (n = 24), seropositivity only to L. chagasi (n = 31), and seropositivity to T. gondii and N. caninum (n = 16). Uninfected dogs were used as control (n = 10). Results: Inflammatory lesions, characterised by mononuclear cell accumulation, composed mainly of CD3+ T lymphocytes predominated in several encephalic regions of the dogs from all the three infected groups, with no difference among them (P = 0.0004), whereas CD79α+ B lymphocytes were detected in very small intensity and presented no difference among groups (P = 0.5313). Furthermore, no association among diseases was detected at the serological enquire. Conclusions: We demonstrate that the peripheral infection by L. chagasi per se can promote the influx of lymphocytes within the nervous milieu as occurs during Toxoplasma and Neospora infections, and the concomitant seropositivity against these pathogens does not exacerbate the inflammatory brain lesions. Therefore, these findings give additional support that the brain should be included in the list of organs affected by visceral leishmaniasis and that even asymptomatic infected dogs may develop brain lesions. © 2013 Sakamoto et al.; licensee BioMed Central Ltd.