Full-thickness tissue engineered oral mucosa for genitourinary reconstruction: A comparison of different collagen-based biodegradable membranes.

Tissue engineering is a method of growing importance regarding clinical application in the genitourinary region. One of the key factors in successfully development of an artificially tissue engineered mucosa equivalent (TEOM) is the optimal choice of the scaffold. Collagen scaffolds are regarded as gold standard in dermal tissue reconstruction. Four distinct collagen scaffolds were evaluated for the ability to support the development of an organotypical tissue architecture. TEOMs were established by seeding cocultures of primary oral epithelial cells and fibroblasts on four distinct collagen membranes. Cell viability was assessed by MTT-assay. The 3D architecture and functionality of the tissue engineered oral mucosa equivalents were evaluated by confocal laser-scanning microscopy and immunostaining. Cell viability was reduced on the TissuFoil E® membrane. A multi-stratified epithelial layer was established on all four materials, however the TEOMs on the Bio-Gide® scaffold showed the best fibroblast differentiation, secretion of tenascin and fibroblast migration into the membrane. The TEOMs generated on Bio-Gide® scaffold exhibited the optimal cellular organization into a cellular 3D network. Thus, the Bio-Gide® scaffold is a suitable matrix for engineering of mucosa substitutes in vitro.

DNA persistence of bite marks on food and its relevance for STR typing.

In forensic DNA analysis, salivary traces at crime scenes are a promising way to identify a person. However, crime scenes are oftentimes investigated a while after the crime and recovered samples might have been degraded leading to poor PCR amplification. Probably due to decomposition and negative visual impression of spoiled food, bite mark samples make up only a small part of our casework routine. In this study, bite marks on apples and chocolate bars as well as on an inert surface (microscope slide) were stored up to 3 weeks indoors and outdoors during different seasons and analyzed for amylase activity and DNA quantity and quality. The results underlined the stability of human nuclear DNA not only on inert but also on biological surfaces and their forensic usefulness even when bite marks are stored 21 days under adverse but realistic conditions at a crime scene. Overall, amylase activity as well as DNA quantity decreased over time depending on storage environment with a certain inter- and intrapersonal variation. But amylase activity testing was not found to be an appropriate screening tool for further analysis. Apple bite marks resulted in generally higher DNA amounts than chocolate bars and microscope slides. Although mold reduced the DNA quantity, complete STR profiles could be analyzed. High air humidity and cold temperatures were found to act preservative on raw food with high water content but caused loss of information over time for smooth inert surfaces and hygroscopic foods like sweets. Many factors are involved in the stability of DNA in bite marks and its resulting quality and quantity available for an STR analysis. However, since there was a substantial proportion of informative STR profiles even from bite marks stored for 21 days, the results encourage the analysis of those even if their visual appearance seems unfavorable.

Persistence of touch DNA on burglary-related tools.

Experts are increasingly concerned by issues regarding the activity level of DNA stains. A case from our burglary-related casework pointed out the need for experiments regarding the persistence of DNA when more than one person touched a tool handle. We performed short tandem repeat (STR) analyses for three groups of tools: (1) personal and mock owned tools; (2) tools, which were first "owned" by a first user and then handled in a burglary action by a second user; and (3) tools, which were first owned by a first user and then handled in a moderate action. At least three types of tool handles were included in each of the groups. Every second user handled the tool with and without gloves. In total, 234 samples were analyzed regarding profile completeness of first and second user as well as properties like detectable major profile or mixture attributes. When second users simulated a burglary by using a tool bare handed, we could not detect the first user as major component on their handles but attribute him to the stain in 1/40 cases. When second users broke up the burglary setup using gloves, the first user matched the DNA handle profile in 37% of the cases. Moderate use of mock borrowed tools demonstrated a material-dependent persistence. In total, we observed that the outcome depends mainly on the nature of contact, the handle material, and the user-specific characteristics. This study intends to supplement present knowledge about persistence of touch DNA with a special emphasis on burglary-related cases with two consecutive users and to act as experimental data for an evaluation of the relevance of alleged hypotheses, when such is needed in a court hearing.

Development and characterization of a new 12-plex ChrX miniSTR system.

Short tandem repeat (STR) markers are extensively used for human identification as well as paternity and forensic casework. X-chromosome STR (X-STR) markers are a powerful complementary system especially in deficiency paternity testing. This study presents the development and characterization of a new X-chromosomal short tandem repeat (STR) multiplex using short amplicon (<200 bp). A total of 366 samples from Punjabi population and 346 samples from Sindhi population were typed for 11 X-chromosomal STR markers: DXS101, DXS6789, DXS6793, DXS7132, DXS7423, DXS7424, DXS8378, DXS9902, GATA31E08, GATA172D05, and HPRTB along with sex-typing locus, amelogenin. Each marker showed a high degree of polymorphism, and the multiplex was sensitive down to 250 pg of human DNA. A total of 78 alleles were found with 5-11 alleles for each marker. The population data can be used as reference database for Sindhi and Punjabi populations.

Comparison of different interpretation strategies for low template DNA mixtures.

DNA typing protocols have been improved over the last few years and even mixtures from minute and low grade DNA stains do not necessarily preclude typing. Nevertheless, in those cases stochastic phenomena tend to hamper interpretation. In order to supplement the current discussion about the interpretation of such challenging data, we focused on different combinations of analyses as an attempt to overcome stochastic problems. We analyzed mixtures of two types of degraded DNA in low template amounts (50 and 100pg DNA per contributor) using four types of multiplex STR typing kits: PowerPlex(®) ESX 17, PowerPlex(®) ESI 17, Investigator(®) ESSplex SE Kit and P11, a non-commercial kit. We employed the results of double or triple analyses for a comparison of different types of interpretation rules based on reporting either only reproducible alleles (consensus interpretation) or all alleles, even if they are only observed once (composite interpretation). The interpreted and composed profiles were compared to the known alleles of the contributors and a "degree of validity" was calculated. When only two single amplifications were taken into account, we observed a higher degree of validity for composite profiles. The difference for consensus interpretation could be compensated when a minimum of three amplifications were carried out. Using the same kit for repeat analyses increases the chances to yield reproducible results required for consensus interpretation. Combining different kits in a complementing approach, on the other hand, offers the opportunity to reduce the number of drop-out alleles: differences in amplicon lengths for specific markers between kits can increase the resulting information. In the case of a few amplifications available this effect might only be visible with the composite method. Several markers like SE33 are particularly affected by this. Based on our observations the consensus interpretation method may not reflect the original profile in an optimal way in some special cases like low template, degraded mixture stains. In those cases the composite interpretation could yield more complete results. However, such a composite profile should be used with caution and only for limited purposes. Generally recommending the consensus interpretation thus seems not to be justified: a more differentiated approach appears to be worthwhile, e.g. the amount of drop-outs, the number of replicates, choice and combination of kits and even a marker specific procedure might be taken into account.