Ultrafast stamping by combination of synchronized galvanometer scanning with DOE’s or SLM

The up-scaling of laser micromachining processes with ultrashort pulses is limited due to heat accumulation and shielding effects. Multi beam scanning represents one of the strategies to overcome this drawback. It is in general realized by combining a diffractive beam splitter with a galvanometer scanner. A full synchronization with the laser repetition rate offers new possibilities with minimum thermal impact. We will demonstrate this by means of a multipulse-drilling on the fly process with a regular 5x5 spot pattern having a spot to spot spacing of 160µm. At a repetition rate of 100 kHz and an average power of 16 W we were able to drill more than 2’300 holes/s in a 10µm thick steel foil. We have further extended this technology with a special light modulator for different periodic spot patterns and more complex intensity distributions

Intragenic deletion in the gene encoding L -gulonolactone oxidase causes vitamin C deficiency in pigs

The absence of L-ascorbic acid (L-AA, or AA) synthesis in scurvy-prone organisms, including humans, other primates, guinea pigs, and flying mammals, was traced to the lack of L-gulonolactone oxidase (GULO) activity. GULO is a microsomal enzyme that catalyzes the terminal step in the biosynthesis of L-AA. Clinical cases of scurvy were described in a family of Danish pigs. This trait is controlled by a single autosomal recessive allele designated od (osteogenic disorder). Here we demonstrate that the absence of GULO activity and the associated vitamin C deficiency in od/od pigs is due to the occurrence of a 4.2-kbp deletion in the GULO gene. This deletion includes 77 bp of exon VIII, 398 bp of intron 7 and 3.7 kbp of intron 8, which leads to a frame shift. The mutant protein is truncated to 356 amino acids, but only the first 236 amino acids are identical to the wild-type GULO protein. In addition, the od allele seems to be less expressed in deficient and heterozygous pigs compared with the normal allele in heterozygous and wild-type animals as determined by ribonuclease protection assay. We also developed a DNA-based test for the diagnosis of the deficient allele. However, we failed to identify the mutated allele in other pig population

Milling applications with GHz burst: Investigations concerning the removal rate and machining quality

GHz-bursts were reported to be highly efficient for ultra-short pulse laser ablation compared to single pulses. However, most comparisons were made for a drilling process or the machining of dimples. We investigated GHz-bursts (5.4 GHz) on copper, brass, stainless steel, silicon, zirconium oxide, soda-lime glass and sapphire for surface structuring applications. Inconsistent with the published results neither a higher removal rate, nor an improvement in the machining quality in case of the metals and silicon was observed, in the contrary, a tremendous drop in the specific removal rate of 90% for the metals and 60% for silicon, compared to single pulses, was measured when a 25 pulse burst (maximum of laser system) was applied. The situation differs for zirconium oxide, where only a moderate influence was observed and for soda-lime glass and sapphire where the specific removal rate increased by a factor of 2.3 and 6, when the number of pulses per burst were raised from 1 to 25

Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells

Objective: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering. Methods: EPCs were isolated from 20ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing. Results: Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues. Conclusions: Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac intervention

Evaluation of MTAP and p16 immunohistochemical deficiency as surrogate marker for CDKN2A/B homozygous deletion in gliomas.

Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for CDKN2A/B deletions can be performed by a variety of approaches, including copy number variation (CNV) analysis based on gene array analysis, next generation sequencing (NGS) or fluorescence in situ hybridisation (FISH), but questions remain regarding the accuracy of testing modalities. In this study, we assessed: (1) the utility of S-methyl-5'-thioadenosine phosphorylase (MTAP) and cellular tumour suppressor protein pl61NK4a (p16) immunostainings as surrogate markers for CDKN2A/B HD in gliomas, and (2) the prognostic value of MTAP, across different histological tumour grades and IDH mutation status. One hundred consecutive cases of diffuse and circumscribed gliomas (Cohort 1) were collected, in order to correlate MTAP and p16 expression with the CDKN2A/B status in the CNV plot of each tumour. IDH1 R132H, ATRX and MTAP immunohistochemistry was performed on next generation tissue microarrays (ngTMAs) of 251 diffuse gliomas (Cohort 2) for implementing survival analysis. Complete loss of MTAP and p16 by immunohistochemistry was 100% and 90% sensitive as well as 97% and 89% specific for CDKN2A/B HD, respectively, as identified on CNV plot. Only two cases (2/100) with MTAP and p16 loss of expression did not demonstrate CDKN2A/B HD in CNV plot; however, FISH analysis confirmed the HD for CDKN2A/B. Moreover, MTAP deficiency was associated with shortened survival in IDH-mutant astrocytomas (n=75; median survival 61 vs 137 months; p<0.0001), IDH-mutant oligodendrogliomas (n=59; median survival 41 vs 147 months; p<0.0001) and IDH-wild-type gliomas (n=117; median survival 13 vs 16 months; p=0.011). In conclusion, MTAP immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with CDKN2A/B status, robustness, rapid turnaround time and low costs, and provides significant prognostic value in IDH-mutant astrocytomas and oligodendrogliomas, while p16 should be used cautiously

Assessing minipig compact jawbone quality at the microscale

Preclinical studies often require animal models for in vivo experiments. Particularly in dental research, pig species are extensively used due to their anatomical similarity to humans. However, there is a considerable knowledge gap on the multiscale morphological and mechanical properties of the miniature pigs’ jawbones, which is crucial for implant studies and a direct comparison to human tissue. In the present work, we demonstrate a multimodal framework to assess the jawbone quantity and quality for a minipig animal model that could be further extended to humans. Three minipig genotypes, commonly used in dental research, were examined: Yucatan, G ̈ottingen, and Sinclair. Three animals per genotype were tested. Cortical bone samples were extracted from the premolar region of the mandible, opposite to the teeth growth. Global morphological, compositional, and mechanical properties were assessed using micro-computed tomography (micro-CT) together with Raman spectroscopy and nano- indentation measurements, averaged over the sample area. Local mineral-mechanical relationships were investigated with the site-matched Raman spectroscopy and micropillar compression tests. For this, a novel femtosecond laser ablation protocol was developed, allowing high-throughput micropillar fabrication and testing without exposure to high vacuum. At the global averaged sample level, bone relative mineralization demonstrated a significant difference between the genotypes, which was not observed from the complementary micro-CT measurements. Moreover, bone hardness measured by nanoindentation showed a positive trend with the relative mineralization. For all genotypes, significant differences between the relative mineralization and elastic properties were more pronounced within the osteonal regions of cortical bone. Site-matched micropillar compression and Raman spectroscopy highlighted the differences between the genotypes’ yield stress and mineral to matrix ratios. The methods used at the global level (averaged over sample area) could be potentially correlated to the medical tools used to assess jawbone toughness and morphology in clinics. On the other hand, the local analysis methods can be applied to quantify compressive bone mechanical properties and their relationship to bone mineralization

The receptor locus for Escherichia coli F4ab/F4ac in the pig maps distal to the MUC4 - LMLN region

Enterotoxigenic Escherichia coli (ETEC) with fimbriae of the F4 family are one of the major causes of diarrhea and death among neonatal and young piglets. Bacteria use the F4 fimbriae to adhere to specific receptors expressed on the surface of the enterocytes. F4 fimbriae exist in three different antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13. A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three-generation pedigree including 45 offspring was generated with the aim to use this recombination event to refine the localization of the F4bcR locus. All pigs were phenotyped using the microscopic adhesion test and genotyped for a total of 59 markers. The recombination event was mapped to a 220-kb region between a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest that the locus for F4bcR is located between the LMLN locus and microsatellite S028

Arthrogryposis multiplex congenita (AMC), a hereditary disease in swine, maps to Chromosome 5 by linkage analysis

Arthrogryposis multiplex congenita (AMC), defined as permanent joint contractures present at birth, is one of the most common congenital defects in piglets and other mammals. A genetic form of arthrogryposis was recently identified in Swiss Large White (LW) pigs. The disease is controlled by a single autosomal recessive allele designated as amc. At least 14 LW AI (artificial insemination) boars (about 25% of the Swiss population) are known to be carriers of the amc allele. A total of 219 pigs were used for linkage analysis, including seven founders (F1), three F0, 160 F2, and 49 F3 animals. All founder pigs were full or half sibs. Of the 219 pigs, 41 (18.7%) were found to be affected, while the remaining 178 (81.3%) were healthy. A comprehensive genome scan revealed that microsatellite SW1987 located on pig (Sus scrofa) Chromosome 5 (SSC5), was linked with AMC. Sixteen additional SSC5 microsatellites were selected for further genotyping to generate a multipoint map covering the AMC region. Significant pairwise linkage (LOD > 6.00) was found for AMC and eight marker loci. The order that best fit with the data was SW963-SW1987-SW152-AMC-(SW904, SW1094)-SWR1526-(SWR1974, SW310). AMC was mapped by linkage analysis to the position 92cM, between SW152 and SW904/SW1094, which are located on SSC5 in bands q12-q2

Composition and micromechanical properties of the femoral neck compact bone in relation to patient age, sex and hip fracture occurrence

Current clinical methods of bone health assessment depend to a great extent on bone mineral density (BMD) measurements. However, these methods only act as a proxy for bone strength and are often only carried out after the fracture occurs. Besides BMD, composition and tissue-level mechanical properties are expected to affect the whole bone's strength and toughness. While the elastic properties of the bone extracellular matrix (ECM) have been extensively investigated over the past two decades, there is still limited knowledge of the yield properties and their relationship to composition and architecture. In the present study, morphological, compositional and micropillar compression bone data was collected from patients who underwent hip arthroplasty. Femoral neck samples from 42 patients were collected together with anonymous clinical information about age, sex and primary diagnosis (coxarthrosis or hip fracture). The femoral neck cortex from the inferomedial region was analyzed in a site-matched manner using a combination of micromechanical testing (nanoindentation, micropillar compression) together with micro-CT and quantitative polarized Raman spectroscopy for both morphological and compositional characterization. Mechanical properties, as well as the sample-level mineral density, were constant over age. Only compositional properties demonstrate weak dependence on patient age: decreasing mineral to matrix ratio (p = 0.02, R2 = 0.13, 2.6 % per decade) and increasing amide I sub-peak ratio I~1660/I~1683 (p = 0.04, R2 = 0.11, 1.5 % per decade). The patient's sex and diagnosis did not seem to influence investigated bone properties. A clear zonal dependence between interstitial and osteonal cortical zones was observed for compositional and elastic bone properties (p  200). The proposed classification algorithm together with the output database of bone tissue properties can be used for the future comparison of existing methods to evaluate bone quality as well as to form a better understanding of the mechanisms through which bone tissue is affected by aging or disease