Integrated additive design and manufacturing approach for the bioengineering of bone scaffolds for favorable mechanical and biological properties

Additive manufacturing (AM) presents the possibility of personalized bone scaffolds with unprecedented structural and functional designs. In contrast to earlier conventional design concepts, e.g. raster-angle, a workflow was established to produce scaffolds with triply periodic minimal surface (TPMS) architecture. A core challenge is the realization of such structures using melt-extrusion based 3D printing. This study presents methods for generation of scaffold design files, finite element (FE) analysis of scaffold Young's moduli, AM of scaffolds with polycaprolactone (PCL), and a customized in vitro assay to evaluate cell migration. The reliability of FE analysis when using computer-aided designed models as input may be impeded by anomalies introduced during 3D printing. Using micro-computed tomography reconstructions of printed scaffolds as an input for numerical simulation in comparison to experimentally obtained scaffold Young's moduli showed a moderate trend (R 2 = 0.62). Interestingly, in a preliminary cell migration assay, adipose-derived mesenchymal stromal cells (AdMSC) migrated furthest on PCL scaffolds with Diamond, followed by Gyroid and Schwarz P architectures. A similar trend, but with an accelerated AdMSC migration rate, was observed for PCL scaffolds surface coated with calcium-phosphate-based apatite. We elaborate on the importance of start-to-finish integration of all steps of AM, i.e. design, engineering and manufacturing. Using such a workflow, specific biological and mechanical functionality, e.g. improved regeneration via enhanced cell migration and higher structural integrity, may be realized for scaffolds intended as temporary guiding structures for endogenous tissue regeneration

Computing H/D-Exchange rates of single residues from data of proteolytic fragments

<p>Abstract</p> <p>Background</p> <p>Protein conformation and protein/protein interaction can be elucidated by solution-phase Hydrogen/Deuterium exchange (sHDX) coupled to high-resolution mass analysis of the digested protein or protein complex. In sHDX experiments mutant proteins are compared to wild-type proteins or a ligand is added to the protein and compared to the wild-type protein (or mutant). The number of deuteriums incorporated into the polypeptides generated from the protease digest of the protein is related to the solvent accessibility of amide protons within the original protein construct.</p> <p>Results</p> <p>In this work, sHDX data was collected on a 14.5 T FT-ICR MS. An algorithm was developed based on combinatorial optimization that predicts deuterium exchange with high spatial resolution based on the sHDX data of overlapping proteolytic fragments. Often the algorithm assigns deuterium exchange with single residue resolution.</p> <p>Conclusions</p> <p>With our new method it is possible to automatically determine deuterium exchange with higher spatial resolution than the level of digested fragments.</p

Sample treatment for tissue proteomics in cancer, toxicology, and forensics

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science. This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies. In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution. With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies. Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest