Biomechanical and biochemical assessment of YB-1 expression in A375 melanoma cell line: Exploratory study

Malignant melanoma is the most lethal form of skin cancer. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT). Migratory melanoma cells exhibit two major migration modes: elongated mesenchymal or rounded amoeboid. Using A375 melanoma cell line and the YB-1 knock-out model, we aimed to elucidate biochemical and biomechanical changes in migration signaling pathways in the context of melanoma metastases. We subjected A375 YB-1 knock-out and parental cells to atomic force microscopy (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Our study demonstrated that the constitutive expression of YB-1 in A375 melanoma cell line appears to be closely related to known biomarkers of epithelial-to-mesenchymal transition, nestin, and vimentin, resulting in a stiffer phenotype, as well as a wide array of proteins involved in RNA, ribosomes, and spliceosomes. YB-1 knock-out resulted in nestin depletion and significantly lower vimentin expression, as well as global upregulation of proteins related to the cytoskeleton and migration. YB-1 knock-out cells demonstrated both morphological features and biochemical drivers of mesenchymal/ameboid migration. Melanoma is a highly plastic, adaptable, and aggressive tumor entity, capable of exhibiting characteristics of different migratory modes

Injection of Porcine Adipose Tissue-Derived Stromal Cells by a Novel Waterjet Technology

Previously, we developed a novel, needle-free waterjet (WJ) technology capable of injecting viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we aimed to investigate the effect of WJ technology on cell viability, surface markers, differentiation and attachment capabilities, and biomechanical features. Porcine adipose tissue-derived stromal cells (pADSCs) were isolated, expanded, and injected by WJ technology. Cell attachment assays were employed to investigate cell–matrix interactions. Cell surface molecules were analyzed by flow cytometry. Cells injected by Williams Needle (WN), normal cannula, or not injected cells served as controls. Biomechanical properties were assessed by atomic force microscopy (AFM). pADSCs injected by the WJ were viable (85.9%), proliferated well, and maintained their in vitro adipogenic and osteogenic differentiation capacities. The attachment of pADSCs was not affected by WJ injection and no major changes were noted for cell surface markers. AFM measurements yielded a significant reduction of cellular stiffness after WJ injections (p < 0.001). WJ cell delivery satisfies several key considerations required in a clinical context, including the fast, simple, and reproducible delivery of viable cells. However, the optimization of the WJ device may be necessary to further reduce the effects on the biomechanical properties of cells

Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties

Biomechanical assessment of osteoarthritic articular cartilage and jaw periosteal cells-based bone constructs

Mechanical features influence nearly every aspect of cell biology and function. However, the underlying mechanisms of the role and how mechanical properties and bio-chemical signals are interconnected is not clearly understood. The advent of atomic force microscopy (AFM) provides a powerful tool for quantifying mechanical properties of living cell, typically the elastic modulus (EM). In the present study, a customized AFM approach was applied on load connective tissues to address and investigate two aspects of the musculoskeletal system. The first study investigated the EM changes that occur in the pericellular matrix (PCM) as a function of the cellular pattern reorganization throughout the course of osteoarthritis (OA). The second study investigated the effect of human plate-let lysate (hPL) supplementation on EM and biochemical composition of jaw periosteum derived progenitor cells (JPCs). During osteoarthritis (OA) triggered cartilage degeneration, the chondrocytes in the tissue spatially rearrange from single to double strings, and then to small and finally big clusters. The spatial patterns act as an image-based biomarker for tissue degeneration during OA. In a physiological state, chondrocytes are surrounded by a specialized form of extracellular matrix (ECM) termed the PCM. The PCM, which also dictates the biome-chanical properties of the tissue, is also being progressively degraded throughout the course of OA. The hypothesis of this study was that OA related changes in the cellular organizational patterns (strings, double strings, and clusters) are associated with structural changes of the PCM and with a loss of elastic properties. The biomechanical properties were measured by AFM on specific pattern selected tissue. Biochemical changes of the main components of the PCM (collagen type VI and perlecan) were investigated by pro-tein analysis techniques. The results indicated that there is a significant and stepwise EM decrease alongside each of the cellular pattern rearrangements. At the same time, the ini-tially compact PCM was degraded progressively, losing its structural integrity. The earli-est point with a significant reduction in protein content was at the transition from single strings to small clusters for collagen type VI, and from double strings to small clusters for perlecan. Interestingly, the first significant EM decrease was observed at the transition from single strings to double strings. It must be noted that at this stage, articular cartilage appears macroscopically intact. Both biomechanical properties (EM) as well as biochemi-cal composition (protein content) were the lowest in big clusters. This study is the first to describe the EM as well as structural changes of the PCM in relation to the OA related chondrocyte rearrangement, confirming the role of these patterns as an image-based bi-omarker for early OA events. The hypothesis of the second study was that human plasma lysate (hPL) media en-richment leads to a higher quality of the ECM in JPCs when compared with the standard fetal calf serum (FCS) condition. For this purpose, JPCs cultured with the aforementioned media supplementations were analyzed in two study arms. RAMAN spectroscopy was used for biochemical characterization, and AFM was employed for biomechanical analy-sis. Raman spectroscopic measurements showed significantly higher phosphate to protein ratios and lower carbonate to phosphate ratios under hPL in comparison to FCS culturing. With respect to the ECM collagen maturity, higher ratios of proline to hydroxyproline as well as higher levels of collagen cross-linking were detected in hPL-cultured JPCs. This indicates that hPL induces a higher degree of collagen maturation in JPCs. AFM data showed a significant increase in EM of the ECM under hPL conditions. This study hence demonstrates that hPL media supplementation of JPCs leads to the formation of a higher ECM quality as when compared to the FCS standard settings. In summary, both studies employed AFM-based elasticity measurements to investi-gate biomechanical features of load-bearing tissues. In both studies, the results obtained were significant and provide further insights which may now be fused into existing axi-oms of biochemical processes. Integration of both biomechanical and biochemical features will play a vital role in future scientific endeavors and will serve to establish an in-depth understanding of cellular biology