Wound lesions caused by ear tagging in unweaned calves: assessing the prevalence of wound lesions and identifying risk factors.

Identification of cattle by ear tagging is legally required to ensure traceability. However, studies indicate that ear tagging causes pain-associated physiological and behavioural responses. The wound healing process and prevalence of wound lesions in calves remain mostly unknown. Therefore, this study sought to estimate the prevalence of wound lesions and identify associated risk factors by assessing ear tagging management in unweaned dairy calves. We conducted one field study with single visits to estimate the prevalence of wound lesions and associated risk factors (Study 1, 42 farms, 802 calves) and one follow-up study with repeated visits to assess farmers' view on ear tag management, the relationship between calf health and wound healing, and the development of wound lesions over time (Study 2, five farms, 42 calves). Study 1 comprised a short interview with the farmer (four questions regarding ear tagging). Ear tag position (on or between ridges) and wound lesions were evaluated using a three-level scoring system (1 = no blood, scab, or pus discharge; 2 = incrustation or scab and slight blood or pus discharge; and 3 = heavy purulent discharge, tissue deformation, or both). In Study 2, farmers were interviewed about ear tagging (30 questions), and 10 calves from each farm were assessed on the day of ear tagging and 1, 3, and 6 weeks after tag insertion. Calf health, ear tag position, and wound characteristics were assessed during all visits. Both studies were analysed descriptively, and odds ratios (ORs) for wound lesions in Study 1 were calculated using logistic regression. Of the ears assessed in Study 1, 31.1% showed clinical signs classified as category 2. Score 3 was less common and was found for 6.7% of all ears. Although the highest incidence of wound lesions was found in calves aged 2-4 weeks, wound lesions were also found in calves aged >10 weeks (18.5%). Identified risk factors for wound lesions were small farm size, calf age, single housing, group size, placement of ear tags on ridges, and other ear's score. Individual farmers in Study 2 were able to place ear tags very accurately, although awareness about ear tag lesions appeared to be low among farmers. Sensitising farmers to this issue, implementing routine check-ups of ear tag wounds 2 weeks after insertion, and considering the identified risk factors may reduce animal welfare impairments associated with ear tagging.

Bioinspired calcium phosphate mineralization on Net-Shape-Nonwoven chitosan scaffolds stimulates human bone marrow stromal cell differentiation.

Chitosan fibers were processed using the Net-Shape-Nonwoven (NSN) technique in order to create porous scaffolds which were functionalized in two bioinspired ways: collagen type I coating and unique mineralization with organically modified hydroxyapatite (ormoHAP). While collagen is common to enhance cell attachment on surfaces, the electric-field assisted migration and deposition of ormoHAP on the surface of the NSN-scaffolds is a novel technique which enables sub-micrometer sized mineralization while maintaining the original pore structure. Microscopy revealed fast attachment and morphological adaptation of the cells on both, the pure and the functionalized NSN-scaffolds. Remarkably, the cell number of osteogenically induced hBMSC on ormoHAP-modified NSN-scaffolds increased 3.5-5 fold compared to pure NSN-scaffolds. Osteogenic differentiation of hBMSC/osteoblasts was highest on collagen-functionalized NSN-scaffolds. RT-PCR studies revealed gene expression of ALP, BSP II, and osteocalcin to be high for all NSN-scaffolds. Overall, the NSN-scaffold functionalization with collagen and ormoHAP improved attachment, proliferation, and differentiation of hBMSC and therefore revealed the remarkable potential of their application for the tissue engineering of bone.

Manipulation of osteoclastogenesis: Bioactive multiphasic silica/collagen composites and their effects of surface and degradation products.

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

Electric field-assisted formation of organically modified hydroxyapatite (ormoHAP) spheres in carboxymethylated gelatin gels.

UNLABELLED: A biomimetic strategy was developed in order to prepare organically modified hydroxyapatite (ormoHAP) with spherical shape. The technical approach is based on electric field-assisted migration of calcium ions and phosphate ions into a hydrogel composed of carboxymethylated gelatin. The electric field as well as the carboxymethylation using glucuronic acid (GlcA) significantly accelerates the mineralization process, which makes the process feasible for lab scale production of ormoHAP spheres and probably beyond. A further process was developed for gentle separation of the ormoHAP spheres from the gelatin gel without compromising the morphology of the mineral. The term ormoHAP was chosen since morphological analyses using electron microscopy (SEM, TEM) and element analysis (EDX, FT-IR, XRD) confirmed that carboxymethylated gelatin molecules use to act as organic templates for the formation of nanocrystalline HAP. The hydroxyapatite (HAP) crystals self-organize to form hollow spheres with diameters ranging from 100 to 500nm. The combination of the biocompatible chemical composition and the unique structure of the nanocomposites is considered to be a useful basis for future applications in functionalized degradable biomaterials. STATEMENT OF SIGNIFICANCE: A novel bioinspired mineralization process was developed based on electric field-assisted migration of calcium and phosphate ions into biochemically carboxymethylated gelatin acting as organic template. Advantages over conventional hydroxyapatite include particle size distribution and homogeneity as well as achievable mechanical properties of relevant composites. Moreover, specifically developed calcium ion or phosphate ion release during degradation can be useful to adjust the fate of bone cells in order to manipulate remodeling processes. The hollow structure of the spheres can be useful for embedding drugs in the core, encapsulated by the highly mineralized outer shell. In this way, controlled drug release could be achieved, which enables advanced strategies for threating bone-related diseases, e.g. osteoporosis and multiple myeloma.

Calcium phosphate phases integrated in silica/collagen nanocomposite xerogels enhance the bioactivity and ultimately manipulate the osteoblast/osteoclast ratio in a human co-culture model.

A human co-culture model of osteoblasts and osteoclasts, derived from bone marrow stromal cells and monocytes respectively, was used to characterize the influence of biomaterial modification on the bioactivity and ultimately the ratio of bone-forming to bone-resorbing cells cultivated directly on the surface. Nanocomposites of silica and collagen have been shown to function as skeletal structures in nature and were reproduced in vitro by using a sol-gel approach. The resulting xerogels exhibit a number of features that make it a valuable system for the development of innovative materials for bone substitution applications. In the present study, the incorporation of different calcium phosphate phases in silica/collagen-based gels was demonstrated to enhance the bioactivity of these samples. This ability of the biomaterial to precipitate calcium phosphate on the surface when incubated in simulated body fluids or cell culture medium is generally considered to an advantageous property for bone substitution materials. By co-cultivating human osteoblasts and osteoclasts up to 42 days on the xerogels, we demonstrate that the long-term ratio of these cell types depends on the level of bioactivity of the substrate samples. Biphasic silica/collagen xerogels exhibited comparably low bioactivity but encouraged proliferation of osteoblasts in comparison to osteoclast formation. A balanced ratio of both cell types was detected for moderately bioactive triphasic xerogels with 5% calcium phosphate. However, enhancing the bioactivity of the xerogel samples by increasing the calcium phosphate phase percentage to 20% resulted in a diminished number of osteoblasts in favor of osteoclast formation. Quantitative evaluation was carried out by biochemical methods (calcium, DNA, ALP, TRAP 5b) as well as RT-PCR (ALP, BSP II, OC, RANKL, TRAP, CALCR, VTNR, CTSK), and was supported by confocal laser scanning microscopy (cell nuclei, actin, CD68, TRAP) as well as scanning electron microscopy.

Effect of silica and hydroxyapatite mineralization on the mechanical properties and the biocompatibility of nanocomposite collagen scaffolds.

A recently established materials concept of biomimetic composites based on silica, collagen, and calcium phosphates was adapted for the preparation of porous scaffolds suitable for tissue engineering applications. Mineralization was achieved by directed nucleation of silica on the templating organic phase during a sol-gel process with or without addition of hydroxyapatite. Both mineral phases (25 wt %, individually or combined in equal shares) influenced the scaffold's morphology at the nanoscale. Enhancement of apparent density and compressive strength was similar for silica or hydroxyapatite mineralization; however the stiffening effect of hydroxyapatite was much higher. All scaffold modifications provided proper conditions for adhesion, proliferation, and osteogenic differentiation of human bone marrow stromal cells. The open porosity allowed cells to migrate throughout the scaffolds while maintaining their viability, both confirmed by MTT staining and confocal laser scanning microscopy. Initial cell distributions were graduated due to collagen mineralization, but balanced out over the cultivation time of 28 days. RT-PCR analyses revealed higher gene expression of ALP but lower expression of BSP II and osteocalcin because of collagen mineralization. The results demonstrate that both silica and hydroxyapatite offer comparable possibilities to tailor mechanical properties of collagen-based scaffolds without being detrimental to in vitro biocompatibility.

Development of an osteoblast/osteoclast co-culture derived by human bone marrow stromal cells and human monocytes for biomaterials testing.

The communication of bone-forming osteoblasts and bone-resorbing osteoclasts is a fundamental requirement for balanced bone remodelling. For biomaterial research, development of in vitro models is necessary to investigate this communication. In the present study human bone marrow stromal cells and human monocytes were cultivated in order to differentiate into osteoblasts and osteoclasts, respectively. Finally, a cultivation regime was identified which firstly induces the differentiation of the human bone marrow stromal cells followed by the induction of osteoclastogenesis through the osteoblasts formed--without the external addition of the factors RANKL and M-CSF. As a feedback on osteoblasts enhanced gene expression of BSP II was detected for modifications which facilitated the formation of large multinuclear osteoclasts. Phenotype characterization was performed by biochemical methods (DNA, LDH, ALP, TRAP 5b), gene expression analysis (ALP, BSP II, RANKL, IL-6, VTNR, CTSK, TRAP, OSCAR, CALCR) as well as light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. After establishing this model on polystyrene, similar positive results were obtained for cultivation on a relevant bone substitution material--a composite xerogel of silica, collagen, and calcium phosphate.

In vitro osteoclastogenesis on textile chitosan scaffold.

Textile chitosan fibre scaffolds were evaluated in terms of interaction with osteoclast-like cells, derived from human primary monocytes. Part of the scaffolds was further modified by coating with fibrillar collagen type I in order to make the surface biocompatible. Monocytes were cultured directly on the scaffolds in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) for up to 18 days. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed the formation of multinuclear osteoclast-like cells on both the raw chitosan fibres and the collagen-coated scaffolds. The modified surface supported the osteoclastogenesis. Differentiation towards the osteoclastic lineage was confirmed by the microscopic detection of cathepsin K, tartrate resistant acid phosphatase (TRAP), acidic compartments using 3-(2,4-dinitroanillino)-3'-amino-N-methyldipropylamine (DAMP), immunological detection of TRAP isoform 5b, and analysis of gene expression of the osteoclastic markers TRAP, cathepsin K, vitronectin receptor, and calcitonin receptor using reverse transcription-polymerase chain reaction (RT-PCR). The feature of the collagen-coated but also of the raw chitosan fibre scaffolds to support attachment and differentiation of human monocytes facilitates cell-induced material resorption--one main requirement for successful bone tissue engineering.

Assessment of anti-inflammatory activity of Poria cocos in sodium lauryl sulphate-induced irritant contact dermatitis.

OBJECTIVE: In the present study, we evaluated the anti-inflammatory activity of Poria cocos (PoCo) on experimentally induced irritant contact dermatitis (ICD) in a repeated sodium lauryl sulphate (SLS) irritation model. METHODS: The anti-irritative effect of PoCo was evaluated with a visual score and quantified by non-invasive bioengineering methods, namely chromametry and transepidermal water loss. Three concentrations of PoCo in base cream DAC (amphiphilic emollient; German pharmacopoeia) were tested in a 4-day repetitive irritation test using SLS. RESULTS: A statistically significant anti-inflammatory activity was observed for PoCo by all three methods when applied in parallel to the induction period of ICD. Application of PoCo after induction of ICD once a day for 5 days, starting just at the end of 4 days, was without any effect. CONCLUSION: An anti-inflammatory efficacy of PoCo on the elicitation phase of the ICD induced by repeated SLS test could be observed and quantified by three independent, non-invasive biophysical assessment parameters. This effect can be explained by its influence on pro-inflammatory enzymes, namely phospholipase A2.

The quality of human skin xenografts on SCID mice: a noninvasive bioengineering approach.

BACKGROUND: Animal models are important tools for studies in skin physiology and pathophysiology. Due to substantial differences in skin characteristics such as thickness and number of adnexa, the results of animal studies cannot always be directly transferred to the human situation. Therefore, transplantation of human skin on to SCID (severe combined immunodeficiency) mice might offer a promising tool to perform studies in viable human skin without the direct need for human volunteers. OBJECTIVES: To characterize the physiological and anatomical changes of a human skin transplant on a SCID animal host. METHODS: In this study human skin was transplanted on to 32 SCID mice and followed for 6 months. Barrier function was assessed by transepidermal water loss (TEWL; tewametry) and moisture content of the stratum corneum was studied by measurement of electrical capacitance (corneometry). RESULTS: The results showed considerable deviations of TEWL values and skin hydration between the grafts and human skin in vivo. The human skin showed epidermal hyperkeratosis and moderate sclerosis of the corium 4 and 6 months after transplantation on to SCID mice. CONCLUSIONS: Our results indicate that human skin does not completely preserve its physiological and morphological properties after transplantation on to SCID mice. Therefore, results from experiments using this model system need to be discussed cautiously.

Intra-articular ganglion cysts of the knee joint: a report of 85 cases and review of the literature.

Intra-articular ganglia and cysts of the knee joint are rare and mostly incidental findings in MRI and arthroscopy. During a period of 15 years, nearly 8000 knees were arthroscopically examined. In total, 85 intra-articular soft tissue masses were found within the knee cavity. Of these, 76 were incidental and asymptomatic findings in arthroscopy performed for treatment of osteoarthritic symptoms. Several repeated minor knee traumata were reported in this group but no histories of serious traumatic events. Nine ganglion cysts were obviously solely responsible for the intermittent or chronic non-specific knee discomfort, and classified as symptomatic. There were no histories of previous injury to the knees, no clinical signs of instabilities or meniscal and femoropatellar pathologies, and no associated further intra-articular lesions in arthroscopy. Forty-nine cystic masses originated from the ACL, 16 from the PCL, 12 from the anterior (eight medial, four lateral) and three from the posterior horn of the menisci (two medial, one lateral). Three were located in the infrapatellar fat pad, one arose from a medial plica and one from a subchondral bone cyst. All ganglion cysts were successfully resected or excised using arthroscopic technique. A review of the literature is given and compared with the findings and data of this study.

Erythema induratum of Bazin and Poncet's disease -- successful treatment with antitubercular drugs.

Erythema induratum of Bazin (EIB) is considered a tuberculide reaction and consists of recurrent painful nodules predominantly on the calves. Clinically it has common features with diseases like nodular vasculitis, perniosis, polyarteritis nodosa and erythema nodosum. Poncet's disease is a reactive arthritis that may accompany tuberculosis. We report a case of a young woman in which the simultaneous occurrence of erythema induratum of Bazin and Poncet's disease led to a clinical picture very similar to Löfgren's syndrome. The final diagnosis was obtained by polymerase chain reaction detection of mycobacterial DNA in a skin biopsy. A systemic therapy with tuberculostatic drugs led to the disappearance of symptoms. The presented case shows the usefulness of polymerase chain reaction diagnostics in EIB patients without other clinical signs of tuberculosis and a confusing combination of symptoms, and further confirms the presence of mycobacterial DNA in EIB lesions.

Efficacy measurement of topical antihistamines: a review.

More and more antihistamines are used in gels or ointments for local antipruritic therapy. Among other factors, the efficacy is dependent on the penetration properties of the respective agents and the optimal choice of vehicle substances. To avoid expensive treatment with unsatisfying success, a reliable efficacy measurement would be desirable prior to the admission of new topical antihistamine preparations. Therefore we reviewed the literature for common methods to assess the efficacy of local antihistamines in healthy volunteers. The principle is to apply the test substance to marked test areas and to challenge the skin after a certain time with a standardised amount of histamine, allergens or mast-cell-degranulating substances. For the test evaluation, the areas of wheal, flare and itch are measured and compared between antihistamine-treated and control fields. Challenge models and most of the described evaluation methods are suited for the preliminary efficacy measurement of antihistamines. However, to be able to compare the results, a standardised procedure used by all investigators would be desirable.

The atopy patch test -- reproducibility and comparison of different evaluation methods.

BACKGROUND: There is still a lack of standardization of the atopy patch test (APT) in test procedures and evaluation methods. Our aim was to examine the reproducibility of APT results and to compare visual evaluation to chromametry and laser Doppler imaging. METHODS: Fifty-two volunteers with atopic eczema/dermatitis syndrome (AEDS) were included. The APT was performed on tape-stripped and unstripped test fields on their backs using cat dander, house dust mite and grass pollen allergens from two different suppliers. Responders were re-tested 4-12 weeks later with the same allergens on their forearms. RESULTS: Using Allergopharma allergens, 14 (26.9%) volunteers showed one or more positive reactions. The reproducibility rate was 56.3%. The Erlangen atopy score in APT-positive and negative volunteers was 19 +/- 6 vs 15 +/- 6. The test agreement in volunteers tested with both allergens, from Allergopharma and Stallergènes, was poor. Correlation of the results between the three evaluation methods was significant (P < or = 0.001). CONCLUSIONS: The low reproducibility rate of APT results and the poor inter-test-agreement using allergens from different suppliers show that much work remains to make the APT a reliable tool in identifying relevant aeroallergens that lead to flare ups of AEDS. Compared to chromametry and laser Doppler imaging, visual scoring was superior in differentiation between irritative and allergic reactions.

Tacrolimus enhances irritation in a 5-day human irritancy in vivo model.

Tacrolimus (FK 506) is a macrolide discovered in 1984 as a metabolic product of Streptomyces tsukabaensis. It has been used successfully in treating atopic dermatitis, allergic contact dermatitis, lichen planus mucosae and pyoderma gangrenosum. In the present study, we evaluated the anti-inflammatory activity of FK 506 in 2 human skin inflammation models. FK 506 as Protopic(R) cream was tested (i) in a 4-day repetitive irritation test with 2 x daily application of sodium lauryl sulphate (SLS), and (ii) in a UVB erythema model. The effect was evaluated visually and quantified by non-invasive bioengineering methods, namely chromametry and tewametry (TEWL). When FK 506 was applied 30 min after SLS irritation, an increased inflammation in comparison to controls was observed with all 3 methods, with only the TEWL data reaching statistical significance. 1 x daily application of FK 506 for 5 days, starting at the end of the 4-day irritation period, was without any effect. Similarly, no effect of FK 506 was seen in the UVB model. In conclusion, FK 506 was shown to enhance experimentally induced irritant contact dermatitis and not to accelerate healing of irritant contact dermatitis and UVB erythema.

[Calculation of 3-D deformity in scoliosis by MRI of the total spine in two perpendicular reconstructed planes]. / Bestimmung des reellen Skoliosewinkels anhand von kernspintomographischen Wirbelsäulenganzaufnahmen im rekonstruierten Coronar- und Sagittalprofil.

AIM: Scoliosis is a spinal deformity that is more complex and does not exist in one plane only. There have been many attempts to analyse three-dimensional spinal deformity, however, these procedures necessitate higher radiation doses. METHOD: In this study we define angles according to the Cobb Definition. By means of trigonometrical evaluation, 3D calculation of spinal deformity is demonstrated using MRI of the total spine in two reconstructed perpendicular planes. 3D spinal analysis was performed on 41 female and 7 male patients with scoliosis. RESULTS: 79 angles were measured by using the Cobb angle in reconstructed coronal plane of MRI of the total spine and, in addition, by using our method. The scoliosis Cobb angles ranged from 11 - 59 degrees (mean: 23 degrees +/- 9 degrees ), the real angles ranged from 12 - 70 degrees (mean: 32 +/- 14 degrees ). There was a poor correlation between Cobb angles and the 3D calculated angles (r = 0.37; p < 0.0001). CONCLUSION: Our method enables us to determine the real angle of scoliosis and to avoid techniques with any radiation risk for the patient.

Probiotic Lactobacillus dose required to restore and maintain a normal vaginal flora.

Forty-two healthy women were randomized to receive one of three encapsulated Lactobacillus rhamnosus GR-1 plus Lactobacillus fermentum RC-14 probiotic dosage regimens or L. rhamnosus GG by mouth each day for 28 days. However, the vaginal flora, assessed by Nugent scoring, was only normal in 40% of the cases, and 14 patients had asymptomatic bacterial vaginosis. Treatment with L. rhamnosus GR-1/L. fermentum RC-14 once and twice daily correlated with a healthy vaginal flora in up to 90% of patients, and 7/11 patients with bacterial vaginosis converted to normal or intermediate scores within 1 month. Ingestion of L. rhamnosus GG failed to have an effect. This study confirms the potential efficacy of orally administered lactobacilli as a non-chemotherapeutic means to restore and maintain a normal urogenital flora, and shows that over 10(8) viable organisms per day is the required dose.