Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule–directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)–induced chemokine (interleukin [IL]–8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane–expressed ICAM-1 and of cell culture supernatant–associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.

Demineralized bone matrices (DBMs) are widely used in bone replacement therapy. Bone tissue of either cancellous or cortical origin is decellularized, demineralized, and sterilized during processing, while retaining portions of native organic extracellular matrix (ECM) proteins that regulate cell–matrix interactions during bone repair. The ECM largely accounts for the distinct functions of cortical and cancellous bone. Differences in three-dimensional architecture and matrix density between cancellous and cortical bone may therefore affect ECM proteome signatures and the resulting cellular microenvironment. In this study, ECM proteins were extracted from processed cancellous and cortical allografts at multiple processing steps and analyzed by quantitative mass spectrometry. We identified distinct extractable proteome signatures associated with bone metabolic functions. Cancellous grafts were relatively enriched in proteins associated with inflammatory, coagulative, and immune-related processes, whereas cortical grafts showed higher abundance of structural and matrix-organization-associated proteins. More extensively processed product formats showed fewer significant protein differences between the cortical and cancellous bone type. Within the limitations of pooled donor material and absent functional validation, these findings provide a proteomic framework for future characterization and evaluation of DBM-based allograft products.

Demineralized bone matrix (DBM)-derived allografts retain extracellular matrix (ECM)-associated factors involved in bone repair, but biochemical protein recovery alone may not predict functional pathway activation. We evaluated the HEK-Blue™ TGF-β Reporter Assay as a targeted method for assessing Smad-dependent reporter activity in DBM-derived materials. ECM proteins were extracted from cortical demineralized bone granules (DBG) using guanidine hydrochloride (GuHCl) or urea and quantified after extraction, dialysis, and sterile filtration. Reporter cells were stimulated with extracted ECM proteins or directly with processed cortical and cancellous products, including DBG, wet heat-treated DBG formulated as Putty (PHT), and gamma-irradiated PHT (PGI). Non-pooled DBM sponge samples were also tested. Secreted embryonic alkaline phosphatase (SEAP) activity served as the functional reporter readout. Material-only no-cell controls assessed material-derived background. PrestoBlue™ readouts served as exploratory quality controls. The urea-derived extract yielded a higher apparent BCA-detectable protein concentration than the GuHCl-derived extract. Only the GuHCl-derived extract induced increasing SEAP activity at matched protein input. Direct stimulation showed reporter activation above the TNF-α pathway-negative cytokine control for cortical DBG, PHT, PGI, and cancellous DBG. DBM sponges also induced detectable SEAP activity. These findings support targeted functional screening of Smad-dependent TGF-β reporter activation in DBM-derived materials, but not total osteoinductivity assessment.