Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs

OBJECTIVE: The purpose was to qualitatively and quantitatively compare the bone formation and graft resorption of two different bone substitutes used in both orthopedic and oral surgery, with autogenous bone as a positive control. MATERIALS AND METHODS: Three standardized bone defects were prepared in both mandibular angles of 12 adult minipigs. The defects were grafted with either autograft, anorganic bovine bone (ABB), or synthetic beta-tricalcium phosphate (beta-TCP). Sacrifice was performed after 1, 2, 4, and 8 weeks for histologic and histomorphometric analysis. RESULTS: At 2 weeks, more new bone formation was seen in defects filled with autograft than with ABB (P approximately 0.0005) and beta-TCP (P approximately 0.002). After 4 weeks, there was no significant difference between beta-TCP and the two other materials. Defects grafted with ABB still exhibited less bone formation as compared with autograft (P approximately 0.004). At 8 weeks, more bone formation was observed in defects grafted with autograft (P approximately 0.003) and beta-TCP (P approximately 0.00004) than with ABB. No difference could be demonstrated between beta-TCP and autograft. beta-TCP resorbed almost completely over 8 weeks, whereas ABB remained stable. CONCLUSION: Both bone substitutes seemed to decelerate bone regeneration in the early healing phase as compared with autograft. All defects ultimately regenerated with newly formed bone and a developing bone marrow. The grafting materials showed complete osseous integration. Both bone substitutes may have a place in reconstructive surgery where different clinical indications require differences in biodegradability.

Early resuscitation with polymerized bovine hemoglobin reverses acidosis, but not peripheral tissue oxygenation, in a severe hamster shock model

Awake hamsters equipped with the dorsal window chamber preparation were subjected to hemorrhage of 50% of the estimated blood volume. Initial resuscitation (25% of estimated blood volume) with polymerized bovine hemoglobin (PBH) or 10% hydroxyethyl starch (HES) occurred in concert with an equivolumetric bleeding to simulate the early, prehospital setting (exchange transfusion). Resuscitation (25% of estimated blood volume) without bleeding was performed with PBH, HES, or autologous red blood cells (HES-RBCs). Peripheral microcirculation, tissue oxygenation, and systemic hemodynamic and blood gas parameters were assessed. After exchange transfusion, base deficit was -8.6 +/- 3.7 mmol/L (PBH) and -5.1 +/- 5.3 mmol/L (HES) (not significant). Functional capillary density was 17% +/- 6% of baseline (PBH) and 31% +/- 11% (HES) (P < 0.05) and arteriolar diameter 73% +/- 3% of baseline (PBH) and 90% + 5% (HES) (P < 0.01). At the end, hemoglobin levels were 3.7 +/- 0.3 g/dL with HES, 8.2 +/- 0.6 g/dL with PBH, and 10.4 +/- 0.8 g/dL with HES-RBCs (P < 0.01 HES vs. PBH and HES-RBCs, P < 0.05 PBH vs. HES-RBCs). Base excess was restored to baseline with PBH and HES-RBCs, but not with HES (P < 0.05). Functional capillary density was 46% +/- 5% of baseline (PBH), 62% + 20% (HES-RBCs), and 36% +/- 19% (HES) (P < 0.01 HES-RBCs vs. HES). Peripheral oxygen delivery and consumption was highest with HES-RBCs, followed by PBH (P < 0.05 HES-RBCs vs. PBH, P < 0.01 HES-RBCs and PBH vs. HES). In conclusion, the PBH led to a correction of base deficit comparable to blood transfusion. However, oxygenation of the peripheral tissue was inferior with PBH. This was attributed to its negative impact on the peripheral microcirculation caused by arteriolar vasoconstriction.

The effect of enamel matrix proteins and deproteinized bovine bone mineral on heterotopic bone formation

To evaluate the osteoinductive potential of deproteinized bovine bone mineral (DBBM) and an enamel matrix derivative (EMD) in the muscle of rats. Sixteen rats were used in this study. The animals were divided in three groups. Group A: a pouch was created in one of the pectoralis profundis muscles of the thorax of the rats and DBBM particles (Bio-Oss) were placed into the pouch. Healing: 60 days. Group B: a small pouch was created on both pectoralis profundis muscles at each side of the thorax midline. In one side, a mixture of EMD (Emdogain) mixed with DBBM was placed into one of the pouches, whereas in the contralateral side of the thorax the pouch was implanted with DBBM mixed with the propylene glycol alginate (PGA--carrier for enamel matrix proteins of EMD). Healing: 60 days. Group C: the same procedure as group B, but with a healing period of 120 days. Qualitative histological analysis of the results was performed. At 60 days, the histological appearance of the DBBM particles implanted alone was similar to that of the particles implanted together with EMD or PGA at both 60 and 120 days. The DBBM particles were encapsulated into a connective tissue stroma and an inflammatory infiltrate. At 120 days, the DBBM particles implanted together with EMD or PGA exhibited the presence of resorption lacunae in some cases. Intramuscular bone formation was not encountered in any group. The implantation of DBBM particles alone, combined with EMD or its carrier (PGA) failed to exhibit extraskeletal, bone-inductive properties.

Guided tissue regeneration/deproteinized bovine bone mineral or papilla preservation flaps alone for treatment of intrabony defects. II: radiographic predictors and outcomes

OBJECTIVES: This study reports the secondary analysis of a randomized-controlled clinical trial designed to assess the efficacy of deproteinized bovine mineral and a collagen membrane in the treatment of intrabony defects. The specific aims of this report are (1) to analyse the radiographic bone changes 1 year after therapy and (2) to assess the association between radiographic defect angle and treatment outcomes. MATERIALS AND METHODS: Baseline and 12-month radiographs were collected from 120 patients with advanced chronic periodontitis from 10 centres in seven countries as part of a multi-centre clinical trial. All patients had at least one intrabony defect > or =3 mm in depth. The treatment consisted of simplified or modified papilla preservation flaps to access the defect. After debridement of the area, a deproteinized bovine mineral and a collagen membrane were applied in the test subjects, and omitted in the controls. Main outcome measures were radiographic bone fill and defect resolution 1 year after surgery. RESULTS: One hundred and twenty pairs of radiographs were obtained, of which 110 pairs were measurable (57 tests and 53 controls). One year after treatment, radiographic resolution of the intrabony component was significantly higher in the test group (3.2+/-1.7 mm) when compared with the controls (1.7+/-1.9 mm). Multivariate analysis indicated that the treatment and the baseline radiographic depth of the intrabony defect significantly influenced the radiographic bone fill of the intrabony defect 1 year following treatment. The percentage of resolution of the defect was influenced by the treatment provided and the baseline plaque score. The baseline radiographic defect angle did not show a significant impact on the clinical and radiographic outcomes. CONCLUSIONS: Regenerative periodontal surgery with a deproteinized bovine bone mineral and a collagen membrane offered additional benefits in terms of radiographic resolution of the intrabony defect and predictability of outcomes with respect to papilla preservation flaps alone.

Uptake of bone-related matrix proteins into implanted deproteinized bovine bone mineral

Deproteinized bovine bone mineral (DBBM) (Bio-Oss®, Geistlich-Pharma, Wohlhusen, Switzerland) is widely used as a bone substitute for the preservation or augmentation of bone volume. After implantation near native bone, new bone may form around the DBBM particles. Since DBBM is very resistant to resorption, it will hardly ever be replaced by bone and, therefore, the mechanical stability largely depends on the extent of bridging between the newly formed bone and the DBBM particles. The molecular factors responsible for the deposition of new bone to the DBBM particles have not been determined. The aim of this study was, therefore, to test the hypothesis that DBBM implanted near bone take up bone-related matrix proteins that are involved in cell-matrix interactions. Cylindrical biopsies harvested from tooth extraction sites filled with DBBM particles were fixed in aldehydes, decalcified, and embedded in LR White resin. Thin sections were incubated with antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two bone proteins involved in cell attachment, signaling, and mineralization. High-resolution immunogold labeling was used to examine protein distribution. BSP and OPN were immunodetected in all DBBM particles and yielded an identical distribution pattern. Most gold particles were found over the peripheral DBBM matrix, although some peripheral regions lacked immunolabeling. The bulk of the interior DBBM portion was mainly free of labeling with the exception of the peripheral matrix of some osteocyte lacunae and canaliculi. It is concluded that DBBM selectively takes up at least BSP and OPN after its implantation at a bone site. BSP and OPN or other molecules accommodating in DBBM may modulate events associated with cell attachment and differentiation.

The locus for bovine dilated cardiomyopathy maps to chromosome 18

Bovine dilated cardiomyopathy (BDCMP) is a severe and terminal disease of the heart muscle observed in Holstein-Friesian cattle over the last 30 years. There is strong evidence for an autosomal recessive mode of inheritance for BDCMP. The objective of this study was to genetically map BDCMP, with the ultimate goal of identifying the causative mutation. A whole-genome scan using 199 microsatellite markers and one SNP revealed an assignment of BDCMP to BTA18. Fine-mapping on BTA18 refined the candidate region to the MSBDCMP06-BMS2785 interval. The interval containing the BDCMP locus was confirmed by multipoint linkage analysis using the software loki. The interval is about 6.7 Mb on the bovine genome sequence (Btau 3.1). The corresponding region of HSA19 is very gene-rich and contains roughly 200 genes. Although telomeric of the marker interval, TNNI3 is a possible positional and a functional candidate for BDCMP given its involvement in a human form of dilated cardiomyopathy. Sequence analysis of TNNI3 in cattle revealed no mutation in the coding sequence, but there was a G-to-A transition in intron 6 (AJ842179:c.378+315G>A). The analysis of this SNP using the study's BDCMP pedigree did not conclusively exclude TNNI3 as a candidate gene for BDCMP. Considering the high density of genes on the homologous region of HSA19, further refinement of the interval on BTA18 containing the BDCMP locus is needed.

Neuropathological and molecular comparison between clinical and asymptomatic bovine spongiform encephalopathy cases

Interest in the proper neuropathological and molecular characterization of bovine spongiform encephalopathy (BSE) has increased since asymptomatic and atypical cases were detected in the cattle population by active disease surveillance. In this respect we investigated a total of 95 confirmed BSE cases originating from different active and passive surveillance categories (clinical suspects, emergency-slaughter, fallen stock and routinely slaughter) in Switzerland for their neuropathological and molecular phenotype. We looked for measurable differences between these categories in lesion profile, severity of spongiform change, degree of astrocytosis as well as immunohistochemical and molecular patterns of the disease-associated isoform of the prion protein (PrPd) in the caudal brainstem. Our results indicate significantly higher intensities of spongiform change in clinically affected compared to asymptomatic BSE cases. Similar effects were in trend observed for the intensities of PrPd deposition and astrocytosis, whereas the frequencies of morphological PrPd types and the molecular patterns in Western immunoblot were not different. Importantly, none of the animals included in this study revealed features of atypical BSE. Taken together, this study suggests that both clinically affected as well as asymptomatic Swiss BSE cases in cattle share the neuropathological and molecular phenotype of classical BSE and that asymptomatic classical BSE cases are at a pre-clinical stage of the disease rather than representing a true sub-clinical form of BSE.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Effect of feeding cows genetically modified maize on the bacterial community in the bovine rumen

Rumen-cannulated cows (n = 4) were fed successively silage made from either conventional or genetically modified (GM) maize. Results revealed no effects of GM maize on the dynamics of six ruminal bacterial strains (investigated by real-time PCR) compared to the conventional maize silage.

Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

Besnoitia besnoiti, an apicomplexan protozoan parasite, is the causative agent of bovine besnoitiosis. This infection may dramatically affect body condition, and, in males, lead to irreversible infertility. While identification of clinical cases and their histopathological confirmation is relatively simple to carry out, finding subclinical forms of infection is more difficult, thus a more sensitive test for the identification of the etiological agent may be an appropriate diagnostic tool. We have developed the ITS1 rDNA-sequence-based conventional and real-time PCR which are highly sensitive and specific for the detection of B. besnoiti infection in cattle. A recombinant internal positive control was introduced to assess possible sample-related inhibitory effects during the amplification reaction and, in order to prevent false-positive results, a pre-PCR treatment of potentially contaminating dU-containing PCR product with uracil-DNA-glycosylase (UDG) was followed.

Quantitation of Guggenheimella bovis and treponemes in bovine tissues related to digital dermatitis

Digital dermatitis is an inflammation of uncertain aetiology in the skin of the foot of cattle. In 2005, a novel microorganism, Guggenheimella bovis, was isolated from the advancing front of digital dermatitis lesions, suggesting a possible role in pathogenesis. In the present study, tissue samples of 20 affected cows were examined by quantitative PCR for G. bovis, treponemes and the total eubacterial load. High numbers of eubacteria and treponemes were found in most lesions, whereas only a few lesions contained Guggenheimella, and only at low concentrations. The results argue against the relevance of G. bovis in the aetiology of digital dermatitis in cattle, but are consistent with a role for treponemes.

[Herd problem: udder health. Retrospective study of farms assessed by the Swiss Bovine Health Service (BHS) from 1999 to 2004]

Data from 59 farms with complaints of udder health problems and insufficient quality of delivered milk that had been assessed by the Swiss Bovine Health Service (BHS) between 1999 and 2004 were retrospectively analysed. Data evaluated included farm characteristics such as farm size, herd size, average milk yield, milking system and housing system, deficits of the milking equipment and the milking practices, and bacteriological results of milk samples from all cows in lactation. The average size of the farms assessed by the BHS was larger than the size of the were evaluated, 42 showed obvious failures which the farm managers could have noticed. Only 5 of the 57 milkers carried out their work according to the generally valid guidelines of the National Mastitis Council. More than 2 basic mistakes were observed in the milking practices of 36 milkers. In 51 farms, mixed infections with several problem bacteria (those present in at least 20 % of the tested cows on a farm) were found. Staphylococcus aureus proved to be the most common problem germ. As the bacteria responsible for the herd problem (the sole problem bacteria detectable on a particular farm) Staphylococcus aureus was detected in 4 farms. The current study revealed that education in the area of milking techniques and milking practices of farmers should be improved in order to reduce the incidence of udder health problems on herd level. Staphylococcus aureus is the most important problem bacteria involved in herds with udder health problems in Switzerland. Staphylococcus aureus might be used in practice as the indicator germ for early recognition of management problems in dairy farms.

Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk

Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 microL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.