A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation.

The analysis of postmortem protein degradation has become of large interest for the estimation of the postmortem interval (PMI). Although several techniques have been published in recent years, protein degradation-based techniques still largely did not exceed basic research stages. Reasons include impractical and complex sampling procedures, as well as highly variable protocols in the literature, making it difficult to compare results. Following a three-step procedure, this study aimed to establish an easily replicable standardized procedure for sampling and processing, and further investigated the reliability and limitations for routine application. Initially, sampling and processing were optimized using a rat animal model. In a second step, the possible influences of sample handling and storage on postmortem protein degradation dynamics were assessed on a specifically developed human extracorporeal degradation model. Finally, the practical application was simulated by the collection of tissue in three European forensic institutes and an international transfer to our forensic laboratory, where the samples were processed and analyzed according to the established protocol.

Morphology of direct and indirect rib fractures.

Rib fractures are a common finding in legal medicine and information on the impact mechanism is relevant for trauma reconstruction. This study focuses on morphological characteristics of rib fractures resulting from direct or indirect force. Fresh human ribs (n = 312) were divided into two groups and broken through local force (direct) and bending (indirect) in anterolateral areas. The ribs were macerated, visually investigated and the results statistically analysed. The indirect fractures showed a significant larger lateral offset of the internal and external fracture ends while the fracture ends of the direct fractures were more often straight, in line. Also, the morphology of the inner and outer fracture edges was significantly related to fracture type. Direct fractures mostly had rough and jagged inner edges (tension side) and straight, smooth outer edges (compression side), whereas indirect fractures more often showed the characteristics vice versa. The results were more convincing in combination and in ribs from persons aged ≤ 75 years at death. In summary, the direct and indirect rib fractures showed significantly different characteristics regarding orientation and offset of the fracture lines and roughness of the inner and outer fracture edges, which can be helpful to distinguish the traumatizing impact mechanisms in forensic autopsy routine.

Sustained release of rhBMP-2 from microporous tricalciumphosphate using hydrogels as a carrier.

BACKGROUND: Tissue engineering and bone substitutes are subjects of intensive ongoing research. If the healing of bone fractures is delayed, osteoinductive materials that induce mesenchymal stem cells (MSCs) to form bone are necessary. The use of Bone Morphogenetic Protein - 2 is a common means to enhance effectiveness and accelerate the healing process. A delivery system that maintains and releases BMP biological activity in controlled fashion at the surgical site while preventing systemic diffusion (and thereby the risk of undesirable effects by controlling the amount of protein implanted) is essential. In this study, we aimed to test a cylindrical TCP-scaffold (porosity ~ 40 %, mean pore size 5 µm, high interconnectivity) in comparison to BMP-2. Recombinant human BMP-2 was dissolved in different hydrogels as a carrier, namely gelatin and alginate cross-linked with CaCl2-solution, or a solution of GDL and CaCO3. FITC-labeled Protein A was used as a model substance for rhBMP-2 in the pre-trials. For loading, the samples were put in a flow chamber and sealed with silicone rings. Using a directional vacuum, the samples were loaded with the alginate-BMP-2-mixture and the loading success monitored by observing changes in a fluorescent dye (FITC labeled Protein A) under a fluorescence microscope. A fluorescence reader and ELISA were employed to measure the release. Efficacy was determined in cell culture experiments (MG63 cells) via Live-Dead-Assay, FACS, WST-1-Assay, pNPP alkaline phosphatase assay and confocal microscopy. For statistical analysis, we calculated the mean and standard deviation and carried out an analysis of variance. RESULTS: Directional vacuum makes it possible to load nearly 100 % of the interconnected micropores with alginate mixed with rhBMP-2. Using alginate hardened with CaCl2 as a carrier, BMP-2's release can be decelerated significantly longer than with other hydrogels - eg, for over 28 days. The effects on osteoblast-like cells were an increase of the growth rate and expression of alkaline phosphatase while triggering no toxic effect. CONCLUSION: The rhBMP-2-loaded microporous TCP scaffolds possess proliferative and osteoinductive potential. Alginate helps to lower the local growth factor dose below the cytotoxic limit, and allows the release period to be lengthened by at least 28 days.

Novel Method for Loading Microporous Ceramics Bone Grafts by Using a Directional Flow.

The aim of this study was the development of a process for filling the pores of a ß-tricalcium phosphate ceramic with interconnected porosity with an alginate hydrogel. For filling of the ceramics, solutions of alginate hydrogel precursors with suitable viscosity were chosen as determined by rheometry. For loading of the porous ceramics with the gel the samples were placed at the flow chamber and sealed with silicone seals. By using a vacuum induced directional flow, the samples were loaded with alginate solutions. The loading success was controlled by ESEM and fluorescence imaging using a fluorescent dye (FITC) for staining of the gel. After loading of the pores, the alginate is transformed into a hydrogel through crosslinking with CaCl2 solution. The biocompatibility of the obtained composite material was tested with a live dead cell staining by using MG-63 Cells. The loading procedure via vacuum assisted directional flow allowed complete filling of the pores of the ceramics within a few minutes (10 ± 3 min) while loading through simple immersion into the polymer solution or through a conventional vacuum method only gave incomplete filling.