Human peripheral blood mononuclear cells (PBMC) are routinely used in vitro to detect cytokine secretion as part of preclinical screens to delineate agonistic and antagonistic action of therapeutic monoclonal antibodies (mAbs). Preclinical value of standard human PBMC assays to detect cytokine release syndrome (CRS) has been questioned, as they did not predict the "cytokine storm" that occurred when healthy human volunteers were given a CD28-specific super-agonist mAb, TGN1412. In this article, we describe a three-dimensional biomimetic vascular test-bed that can be used as a more physiologically relevant assay for testing therapeutic Abs. For developing such a system, we used TGN1412 as a model mAb. We tested soluble TGN1412 on various combinations of human blood components in a module containing endothelial cells grown on a collagen scaffold and measured cytokine release using multiplex array. Our system, consisting of whole leukocytes, endothelial cells, and 100% autologous platelet-poor plasma (PPP) consistently produced proinflammatory cytokines in response to soluble TGN1412. In addition, other mAb therapeutics known to induce CRS or first infusion reactions, such as OKT3, Campath-1H, or Herceptin, generated cytokine profiles in our model system consistent with their in vivo responses. As a negative control we tested the non-CRS mAbs Avastin and Remicade and found little difference between these mAbs and the placebo control. Our data indicate that this novel assay may have preclinical value for predicting the potential of CRS for mAb therapeutics.
This research was carried out to create a pH-responsive polymeric system for the targeted drug delivery of Diloxanide furoate. It relied on sodium alginate (Na-Alg) and Carbopol 934P as building blocks. Using an aqueous free radical polymerization method, SCH1-SCH12 was created with varying polymer, MAA, and MBA input ratios. Positive outcomes were seen in the swelling and release profiles at higher pH levels. Hydrogel formation, as well as component compatibility, thermal stability, and Diloxanide furoate loading, were all validated by instrumental characterization. A drug loading percentage of 83.56% was determined, with the swelling reaching 743.19%. For the formulation with MBA, the gel fraction was 94.58%. The release of diloxanide furoate increased to 91.77% at neutral pH. The formulation containing Carbopol 934P provided the highest mucoadhesion force (3993.42 dynes/cm2). The created hydrogel has been shown to be biocompatible by toxicological testing of the network. Based on the findings, the created polymeric nexus proved promising for pH-dependent localized and regulated delivery of Diloxanide furoate.
Cadaveric decellularized bone tissue is utilized as an allograft in many musculoskeletal surgical procedures. Typically, the allograft acts as a scaffold to guide tissue regeneration with superior biocompatibility relative to synthetic scaffolds. Traditionally these scaffolds are machined into the required dimensions and shapes. However, the geometrical simplicity and, in some cases, limited dimensions of the donated tissue restrict the use of allograft scaffolds. This could be overcome by additive manufacturing using granulated bone that is both decellularized and demineralized. In this study, the large area projection sintering (LAPS) method is evaluated as a fabrication method to build porous structures composed of granulated cortical bone bound by polycaprolactone (PCL). This additive manufacturing method utilizes visible light to selectively cure the deposited material layer-by-layer to create 3D geometry. First, the spreading behavior of the composite mixtures is evaluated and the conditions to attain improved powder bed density to fabricate the test specimens are determined. The tensile strength of the LAPS fabricated samples in both dry and hydrated states are determined and compared to the demineralized cancellous bone allograft and the heat treated demineralized-bone/PCL mixture in mold. The results indicated that the projection sintered composites of 45–55 wt %. Demineralized bone matrix (DBM) particulates produced strength comparable to processed and demineralized cancellous bone.
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