Scanning Electron Microscopy Appearance Research Articles

A Comparison of Sterilization Techniques for Production of Decellularized Intestine in Mice.

Tissue-engineered small intestinal implants are being widely investigated as a potential treatment for children with short bowel syndrome, yet are currently limited by their growth potential and relatively low surface area. To address this gap in the field, several investigators have utilized whole organ decellularization of the small intestine as a platform for subsequent growth of intestinal tissue. However, such scaffold-cell constructs require sterilization as a prerequisite for implantation, and the effects of the different pathogen-clearance techniques used on the tissue architecture remains unknown. The effects of four different published protocols for pathogen clearance of decellularized intestine, namely 0.1% peracetic acid (PAA), 0.18% PAA +4.8% ethanol (EtOH), 0.08% PAA +1% hydrogen peroxide (H2O2), and ultraviolet (UV) sterilization were compared using qualitative and quantitative techniques to assess changes to the extracellular matrix, cytocompatibility, and biocompatibility. All methods of sterilization of decellularized intestine were found to be equally effective and each method had similar histologic and scanning electron microscopy appearance of the sterilized tissue. In addition, collagen and glycosaminoglycan quantities, and the ability to support cell growth were similar among all methods. This study provides insights into the change in crypt villous architecture of the extracellular matrix with all sterilization techniques studied. Our findings demonstrate that sterilization affects the microarchitecture significantly, which has not been well accounted for in studies to date, and we were unable to identify a single best agent to achieve tissue sterilization while preserving the microarchitectural features of the tissue.

Sickled Erythrocyte Morphology in the Bone Marrow of Patients with Sickle Cell Disease: Previously Unrecognized Forms of Sickled Erythrocytes May Have a Pathogenetic Role in Bone Pain of Painful Crises.

Background: The painful crises of sickle cell disease (SCD) are characteristically localized to the skeleton. Various lines of evidence suggest that bone and bone marrow (BM) infarction contribute to the pathophysiology of such pain in which sickled red blood cells (SRBC) are believed to play a part. However, there is little information about the morphological characteristics and role of SRBC in BM during the crises or indeed, in the “steady state”. Occasional light microscopic observations in the BM of SCD patients show numerous markedly distorted cells, presumed to be SRBC, types of which have not been previously reported from this site or the peripheral blood (PB). We therefore studied a series of BM biopsies from SCD patients to determine whether such changes are consistently present.Methods: We microscopically reviewed archival BM biopsies from SCD patients for abnormally shaped RBC and compared the findings with SRBC on Wright-stained smears of PB collected simultaneously with the biopsies. The material consisted of Wright-Giemsa stained smears of aspirated BM and hematoxylin-eosin stained histological sections of clotted aspirate and / or trephine biopsies. We also examined fresh BM RBC by scanning and transmission electron microscopy (EM). We determined the relationship between the percentage of SRBC in BM and PB on the one hand, and fetal hemoglobin (HbF) levels on the other.Results: We studied the BM of 43 SCD patients. By LM, we observed markedly distorted RBC in the BM of the majority of patients who also had BM SRBC that were more typical forms. The distorted forms included vastly elongated erythrocytes, measuring up to 40 microns in length and less than 1 micron in width, some fixed into rod like structures, but others as sinuous, snake-like forms that have not been previously described. Scanning EM appearances also confirmed the features observed by LM. By contrast, none of PB samples showed similarly distorted SRBC. Although the percentage of typical SRBC in BM was concordant with that in the PB (p<0.001) SRBC numbers in both PB and BM were inversely proportional to the level of HbF.(Table 1)Conclusion: We conclude that the proportions of markedly distorted SRBC present in the BM of SCD patients are inversely proportional to HbF level and may thus play a pathogenetic role in the localization of severe pain to the skeleton during painful crises.Table 1Correlations between sickled RBC estimates and blood fetal hemoglobin levelsMSRBC MARROWMBMSRBCMSRBCLOT+TREPHINEMSRBC PBMHbFMHbFMHbFMHbFr−0.3806−0.3293−0.2685−0.384p< 0.05< 0.05NS< 0.05MSRBC= Mean SRBC marrow and peripheral blood together. MBMSRBC = Mean SRBC in bone marrow aspirates only. MSRBCLOT+TREPHINE = Mean SRBC, clotted marrow aspirate and trephine together. MSRBCPB = Mean SRBC in peripheral blood. MHbF = Mean HbF level in blood. r = correlation coefficient. NS = Not significant.

The hardness locus in Australian wheat lines

The genetic factors that determine grain hardness in Australian wheat (Triticum aestivum L.) germplasm were investigated by studying the grain from 4 crosses (160–180 lines per cross). Although not all the crosses were between hard and soft wheats, the doubled haploid lines derived from the crosses showed significant variation in hardness as assessed either by the Single-Kernel Characterisation System (SKCS 4100) or the scanning electron microscopy appearance of cut surfaces. The wheat cultivars used in the study were Cranbrook, Halberd, CD87, Katepwa, Sunco, Tasman, Egret, and Sunstar and of these only Egret is normally regarded as soft. The quantitative information from the SKCS 4100 was integrated into the genetic map information established for the Cranbrook Halberd, CD87 Katepwa, and Sunco Tasman crosses. For the Egret Sunstar cross, the limited information available from the positioning of genetic markers was used to specifically examine the linkage between the hardness trait and the Pina-D1 (puroindoline) genetic locus on 5DS, and very close linkage was established. The Egret Sunstar cross was also used to develop a more rigorous rheologically based analysis of the raw SKCS crush response data. In addition, the cut surface of the lines was analysed and most (98%) of the samples showed a genetic linkage between the appearance of the cut surface (related to vitreousness) and the SKCS hardness index. Among the other crosses only Cranbrook Halberd showed linkage of the hardness trait to the previously identified hardness locus (ha) located on 5DS as defined by DNA markers for the Pina-D1 locus, and the microsatellite wmc233. The statistical association was shown to be highly significant, with approximately 30% of the variation accounted for by the 5DS region. Another region on chromosome 4D showed a significant association in the Cranbrook Halberd cross. The CD87 Katepwa cross did not show any consistent associations between the SKCS measures and chromosome region, whereas in the Sunco Tasman cross a highly significant association only on chromosome 4B (accounting for 20% of the variation) was suggested. The Sunco Tasman cross showed an overlap of the chromosome region that accounted for variation in both grain weight and hardness and this influence of grain weight on hardness was independently confirmed by a detailed qualitative rheological analysis of the crush response profiles for the Egret Sunstar lines. It is evident from the study that, in Australian wheat lines, there are some major effects on grain hardness that are not associated with the classical ha locus located on 5DS.