Development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of antibodies against fowl adenovirus serotypes 1 and 4 in chickens.

Conventional serological methods for detection and differentiation of antibodies against fowl aviadenoviruses (FAdVs) are laborious and time-consuming, therefore ELISAs based upon recombinant proteins were developed in the present study to overcome this limitation for clinically relevant serotypes FAdV-1 and FAdV-4. In order to develop serotype-specific ELISAs, the two distinct fibers, fiber-1 (fib-1) and fiber-2 (fib-2), characteristically present only in FAdV-1 and FAdV-4, were applied separately as coating antigens. Sera raised against each recombinant fib-1 and fib-2 of FAdV-1 and FAdV-4 did not react with any of the heterologous fiber ELISAs, as anticipated by the low degree of amino acid identity between those FAdV fibers (23.1-41.2%), indicating that heterologous fibers do not share common epitopes. Testing of 172 monospecific sera, raised against all FAdV serotypes (1-8a and 8b-11), retrieved specificities between 99.3% and 100.0% for the ELISAs, further substantiating the serotype-specificity of fibers. Investigating sera from chickens experimentally inoculated with different FAdV-1 or FAdV-4 strains revealed that ELISAs were equally or more sensitive than the virus-neutralization (VN) test. Furthermore, strong correlations were demonstrated between fiber antibody titres and neutralization activity. Particularly, sera directed against live virus showed a pronounced fiber antibody response, which might be explained by an excessive production of fibers during infection. Application of the newly developed fiber ELISAs on field sera with heterogeneous serological status demonstrated high sensitivity and serotype-specificity of this test system, providing for the first time a diagnostic tool for mass screening of chicken flocks against FAdV serotypes, namely FAdV-1 and FAdV-4.

In Vivo Investigation of 3D-Printed Calcium Magnesium Phosphate Wedges in Partial Load Defects.

Bone substitutes are ideally biocompatible, osteoconductive, degradable and defect-specific and provide mechanical stability. Magnesium phosphate cements (MPCs) offer high initial stability and faster degradation compared to the well-researched calcium phosphate cements (CPCs). Calcium magnesium phosphate cements (CMPCs) should combine the properties of both and have so far shown promising results. The present study aimed to investigate and compare the degradation and osseointegration behavior of 3D powder-printed wedges of CMPC and MPC in vivo. The wedges were post-treated with phosphoric acid (CMPC) and diammonium hydrogen phosphate (MPC) and implanted in a partially loaded defect model in the proximal rabbit tibia. The evaluation included clinical, in vivo µ-CT and X-ray examinations, histology, energy dispersive X-ray analysis (EDX) and scanning electron microscopy (SEM) for up to 30 weeks. SEM analysis revealed a zone of unreacted material in the MPC, indicating the need to optimize the manufacturing and post-treatment process. However, all materials showed excellent biocompatibility and mechanical stability. After 24 weeks, they were almost completely degraded. The slower degradation rate of the CMPC corresponded more favorably to the bone growth rate compared to the MPC. Due to the promising results of the CMPC in this study, it should be further investigated, for example in defect models with higher load.

In vivo investigation of open-pored magnesium scaffolds LAE442 with different coatings in an open wedge defect.

The magnesium alloy LAE442 showed promising results as a bone substitute in numerous studies in non-weight bearing bone defects. This study aimed to investigate the in vivo behavior of wedge-shaped open-pored LAE442 scaffolds modified with two different coatings (magnesium fluoride (MgF2, group 1)) or magnesium fluoride/calcium phosphate (MgF2/CaP, group 2)) in a partial weight-bearing rabbit tibia defect model. The implantation of the scaffolds was performed as an open wedge corrective osteotomy in the tibia of 40 rabbits and followed for observation periods of 6, 12, 24, and 36 weeks. Radiological and microcomputed tomographic examinations were performed in vivo. X-ray microscopic, histological, histomorphometric, and SEM/EDS analyses were performed at the end of each time period. µCT measurements and X-ray microscopy showed a slight decrease in volume and density of the scaffolds of both coatings. Histologically, endosteal and periosteal callus formation with good bridging and stabilization of the osteotomy gap and ingrowth of bone into the scaffold was seen. The MgF2 coating favored better bridging of the osteotomy gap and more bone-scaffold contacts, especially at later examination time points. Overall, the scaffolds of both coatings met the requirement to withstand the loads after an open wedge corrective osteotomy of the proximal rabbit tibia. However, in addition to the inhomogeneous degradation behavior of individual scaffolds, an accumulation of gas appeared, so the scaffold material should be revised again regarding size dimension and composition.

Comparison of degradation behavior and osseointegration of 3D powder-printed calcium magnesium phosphate cement scaffolds with alkaline or acid post-treatment.

Due to the positive effects of magnesium substitution on the mechanical properties and the degradation rate of the clinically well-established calcium phosphate cements (CPCs), calcium magnesium phosphate cements (CMPCs) are increasingly being researched as bone substitutes. A post-treatment alters the materials' physical properties and chemical composition, reinforcing the structure and modifying the degradation rate. By alkaline post-treatment with diammonium hydrogen phosphate (DAHP, (NH4)2HPO4), the precipitation product struvite is formed, while post-treatment with an acidic phosphate solution [e.g., phosphoric acid (PA, H3PO4)] results in precipitation of newberyite and brushite. However, little research has yet been conducted on newberyite as a bone substitute and PA post-treatment of CMPCs has not been described in the accessible literature so far. Therefore, in the present study, the influence of an alkaline (DAHP) or acid (PA) post-treatment on the biocompatibility, degradation behavior, and osseointegration of cylindrical scaffolds (h = 5.1 mm, Ø = 4.2 mm) produced from the ceramic cement powder Ca0.75Mg2.25(PO4)2 by the advantageous manufacturing technique of three-dimensional (3D) powder printing was investigated in vivo. Scaffolds of the material groups Mg225d (DAHP post-treatment) and Mg225p (PA post-treatment) were implanted into the cancellous part of the lateral femoral condyles in rabbits. They were evaluated up to 24 weeks by regular clinical, X-ray, micro-computed tomographic (µCT), and histological examinations as well as scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analysis and compared with tricalcium phosphate (TCP). All materials showed excellent biocompatibility and rapid osseointegration. While TCP degraded only slightly, the CMPCs showed almost complete degradation. Mg225d demonstrated significantly faster loss of form and demarcability from surrounding bone, scaffold volume reduction, and significantly greater degradation on the side towards the bone marrow than to the cortex than Mg225p. Simultaneously, numerous bone trabeculae have grown into the implantation site. While these were mostly located on the side towards the cortex in Mg225d, they were more evenly distributed in Mg225p and showed almost the same structural characteristics as physiological bone after 24 weeks in Mg225p. Based on these results, the acid post-treated 3D powder-printed Mg225p is a promising degradable bone substitute that should be further investigated.

Effect of pore size on tissue ingrowth and osteoconductivity in biodegradable Mg alloy scaffolds.

Magnesium has mechanical properties similar to those of bone and is being considered as a potential bone substitute. In the present study, two different pore sized scaffolds of the Mg alloy LAE442, coated with magnesium fluoride, were compared. The scaffolds had interconnecting pores of either 400 (p400) or 500 µm (p500). ß-TCP served as control. Ten scaffolds per time group (6, 12, 24, 36 weeks) were implanted in the trochanter major of rabbits. Histological analyses, µCT scans, and SEM/EDX were performed. The scaffolds showed slow volume decreases (week 36 p400: 9.9%; p500: 7.5%), which were accompanied by uncritical gas releases. In contrast, ß-TCP showed accelerated resorption (78.5%) and significantly more new bone inside (18.19 ± 1.47 mm3). Bone fragments grew into p400 (0.17 ± 0.19 mm3) and p500 (0.36 ± 0.26 mm3), reaching the centrally located pores within p500 more frequently. In particular, p400 displayed a more uneven and progressively larger surface area (week 36 p400: 253.22 ± 19.44; p500: 219.19 ± 4.76 mm2). A better osseointegration of p500 was indicated by significantly more trabecular contacts and a 200 µm wide bone matrix being in the process of mineralization and in permanent contact with the scaffold. The number of macrophages and foreign body giant cells were at an acceptable level concerning resorbable biomaterials. In terms of ingrown bone and integrative properties, LAE442 scaffolds could not achieve the results of ß-TCP. In this long-term study, p500 appears to be a biocompatible and more osteoconductive pore size for the Mg alloy LAE442.

Comparative microcomputed tomographic structural analysis of the trabecular and cortical bone architecture of radius and ulna in toy dog breeds.

Radius and ulna fractures can be detected in dogs with an incidence of 18.0% and occur more frequently in toy breeds with increased complication rates. The reasons for the predisposition and the increased complication rate of antebrachial fractures in toy breeds are not yet fully understood. The aim of this study was to research the associated mechanisms by investigating the trabecular structure parameters (BV/TV (%), Tb.N (mm-1), Tb.Th (mm), Tb.Sp (mm), Conn. D (mm-3), DA) and the cortical bone density (Mean Density (mg HA/ccm)) for toy breeds and small dogs of other breeds using microcomputed tomography and to identify the differences between both groups. A total of 70 forearms, originating from toy breeds (n = 36) and small dogs of other breeds (n = 34) were evaluated by microcomputed tomography. The results of this study showed a lower bone volume fraction (BV/TV), more (Tb.N) but thinner trabeculae (Tb.Th) with greater separation (Tb.Sp) and decreased diaphyseal cortical density (Mean Density) in toy breed dogs. This could thus contribute to the increased risk of antebrachial fractures in toy breeds. Within the group of toy breeds inhomogeneous results were observed, whereby the values of the Chihuahuas deviated. This breed had significantly thinner trabeculae (Tb.Th) with a greater trabecular separation (Tb.Sp), lower bone volume fraction (BV/TV) and significantly lower diaphyseal cortical density (Mean Density). These parameters could therefore indicate an increased antebrachial fracture and complication risk, especially in Chihuahuas.

The cat as a small dog?-Comparison of trabecular and cortical bone microarchitecture of radius and ulna in cats and small dogs using microcomputed tomography.

The forearms of dogs and cats do not only differ anatomically from each other, but there are also differences in prevalence of radius and ulna fractures between the two species. The prevalence of antebrachial fractures is 18.0% in dogs and 2.0-8.0% in cats. Many studies focus solely on the trabecular and cortical bone structure of dogs and the characteristics of the cat are often disregarded. The aim of this study was to evaluate the trabecular structure parameters [bone volume fraction per total volume (BV/TV), bone surface per total volume (BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density (Conn. D), degree of anisotropy (DA)] and the diaphyseal cortical bone density (Mean Density) of the antebrachium in cats and small dogs to visualise their differences. For this purpose, a total of 32 forearms of cats (n = 8) and small dogs (n = 8) were evaluated using microcomputed tomography and the findings were compared. The results of the study showed that cats had higher values for BV/TV, Tb.Th, Tb.Sp, DA and Mean Density and lower values for BS/BV, Tb.N and Conn.D at radius and ulna compared to dogs. According to the results of this study, the higher bone volume fraction (BV/TV), thicker trabeculae (Tb.Th), increased anisotropy (DA) and significantly higher diaphyseal cortical density (Mean Density) could contribute to the lower fracture risk of the antebrachium in cats compared to small dogs.

In-Vivo Degradation Behavior and Osseointegration of 3D Powder-Printed Calcium Magnesium Phosphate Cement Scaffolds.

Calcium magnesium phosphate cements (CMPCs) are promising bone substitutes and experience great interest in research. Therefore, in-vivo degradation behavior, osseointegration and biocompatibility of three-dimensional (3D) powder-printed CMPC scaffolds were investigated in the present study. The materials Mg225 (Ca0.75Mg2.25(PO4)2) and Mg225d (Mg225 treated with diammonium hydrogen phosphate (DAHP)) were implanted as cylindrical scaffolds (h = 5 mm, Ø = 3.8 mm) in both lateral femoral condyles in rabbits and compared with tricalcium phosphate (TCP). Treatment with DAHP results in the precipitation of struvite, thus reducing pore size and overall porosity and increasing pressure stability. Over 6 weeks, the scaffolds were evaluated clinically, radiologically, with Micro-Computed Tomography (µCT) and histological examinations. All scaffolds showed excellent biocompatibility. X-ray and in-vivo µCT examinations showed a volume decrease and increasing osseointegration over time. Structure loss and volume decrease were most evident in Mg225. Histologically, all scaffolds degraded centripetally and were completely traversed by new bone, in which the remaining scaffold material was embedded. While after 6 weeks, Mg225d and TCP were still visible as a network, only individual particles of Mg225 were present. Based on these results, Mg225 and Mg225d appear to be promising bone substitutes for various loading situations that should be investigated further.

Comparison of two pore sizes of LAE442 scaffolds and their effect on degradation and osseointegration behavior in the rabbit model.

The magnesium alloy LAE442 emerged as a possible bioresorbable bone substitute over a decade ago. In the present study, using the investment casting process, scaffolds of the Magnesium (Mg) alloy LAE442 with two different and defined pore sizes, which had on average a diameter of 400 µm (p400) and 500 µm (p500), were investigated to evaluate degradation and osseointegration in comparison to a ß-TCP control group. Open-pored scaffolds were implanted in both greater trochanter of rabbits. Ten scaffolds per time group (6, 12, 24, and 36 weeks) and type were analyzed by clinical, radiographic and µ-CT examinations (2D and 3D). None of the scaffolds caused adverse reactions. LAE442 p400 and p500 developed moderate gas accumulation due to the Mg associated in vivo corrosion, which decreased from week 20 for both pore sizes. After 36 weeks, p400 and p500 showed volume decreases of 15.9 and 11.1%, respectively, with homogeneous degradation, whereas ß-TCP lost 74.6% of its initial volume. Compared to p400, osseointegration for p500 was significantly better at week 2 postsurgery due to more frequent bone-scaffold contacts, higher number of trabeculae and higher bone volume in the surrounding area. No further significant differences between the two pore sizes became apparent. However, p500 was close to the values of ß-TCP in terms of bone volume and trabecular number in the scaffold environment, suggesting better osseointegration for the larger pore size.

Fiber-based fluorescent microsphere immunoassay (FMIA) as a novel multiplex serodiagnostic tool for simultaneous detection and differentiation of all clinically relevant fowl adenovirus (FAdV) serotypes.

The recent emergence of fowl aviadenovirus (FAdV) induced disease outbreaks in chicken flocks worldwide, with distinct aetiologies confined to particular FAdV species and serotypes, is increasingly urging the need for specific and mass-applicable antibody screening systems. Despite this exigency, there are to date no available serological procedures which satisfactorily combine the criteria for sensitive detection of antibodies against FAdVs, diagnostic reliability in face of cross-reactions and requirements for a rapid and large-scale application. In order to address this gap, a multiplexed fluorescent microsphere immunoassay (FMIA) based on recombinant FAdV fiber proteins from six different serotypes FAdV-1, -2, -4, -8a, -8b and -11 was developed, which enabled simultaneous detection of antibodies against all clinically relevant serotypes in a single reaction within a high throughput setting. Based on a panel of >300 monospecific antisera raised against each of the 12 FAdV serotypes, 100% serotype-specificity was demonstrated for FAdV-1 (FAdV-A) and FAdV-4 (FAdV-C) fiber-based analytes. Analytes based on serotypes affiliated to FAdV-D and FAdV-E exhibited moderately lower specificities of 91.2-95.7%. This was attributed almost exclusively to mutual recognition between FAdV-2 and -11 field strains and to a much lesser extent to reference strains, supporting earlier proposals to merge them into a single serotype. Similarly, extensive cross-reactions between FAdV-8a and -8b were noted. Altogether intraspecies cross-reactions can be attributed to viruses with a close etiological intersection. Antisera against other important avian viruses remained negative by the FMIA, further validating its specificity. Compared to the virus-neutralization (VN) test, FMIA and individual fiber-based enzyme-linked immunosorbent assays (ELISAs) were equally sensitive in the detection of sera against FAdV-2 and -11, as well as FAdV-8a and -8b field strains, while they were even superior to VN test in detection of FAdV-1 and FAdV-4 responses, likely attributed to a relative abundance of fiber antibodies early upon infection. Moreover, application of the FMIA on field samples comprising a diversified response against all 12 FAdV serotypes further consolidated its specificity and agreement with VN test.