Viable Vitreous Grafts of Whole Porcine Menisci for Transplant in the Knee and Temporomandibular Joints.

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts.

Comparison of 2 self-adhesive resin cements with or without a self-etching primer.

Self-adhesive resin cements that eliminate the primer step have been introduced to simplify the bonding protocol for indirect restorations. The aim of the present study was to compare the shear bond strengths (SBSs) of 2 self-adhesive resin cements used with or without a self-etching primer. The hypothesis was that adding a separate primer component to the self-adhesive systems would increase the SBS at the tooth-adhesive interface. One hundred twenty extracted human molars were hemisectioned and embedded in epoxy. Specimens were polished to expose enamel or dentin surfaces and randomly assigned to 12 test groups (n = 20). The tested variables were (1) the type of bonded tissue (enamel or dentin); (2) the cement used; (3) whether the cement was applied with or without a primer; (4) and whether the primer was air dried or photopolymerized. Bonding jigs were used to apply the self-adhesive resin cement to the tooth surfaces. A 2-minute self-cure was followed by 20 seconds of light curing. Specimens were stored in water for 24 hours and then subjected to SBS testing in a universal testing machine. Fractured specimens were examined under a microscope to determine the modes of failure. Mean SBS values were compared using a paired Student t test (with post hoc Tukey test) and an analysis of variance (α = 0.05). Compared to the control groups, the air-dried primer groups showed SBSs that were 4-6 times greater for the specimens bonded to dentin and 2-3 times greater for specimens bonded to enamel. The photopolymerized primer groups followed the same trend. The photopolymerized groups showed higher percentages of cohesive tooth failure than did air-dried primer groups. Placement of a self-etching primer prior to a self-adhesive resin cement significantly increased the SBS to tooth structure of the "all-in-1" resin cements that were tested. Photopolymerizing the primer did not significantly increase the bond strength.

The Efficacy of the Wee1 Inhibitor MK-1775 Combined with Temozolomide Is Limited by Heterogeneous Distribution across the Blood-Brain Barrier in Glioblastoma.

PURPOSE: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide. EXPERIMENTAL DESIGN: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging. RESULTS: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors. CONCLUSIONS: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM.

Thermodynamics of binding of structurally similar ligands to histone deacetylase 8 sheds light on challenges in the rational design of potent and isozyme-selective inhibitors of the enzyme.

Among the different histone deacetylase (HDAC) isozymes, HDAC8 is the most highly malleable enzyme, and it exhibits the potential to accommodate structurally diverse ligands (albeit with moderate binding affinities) in its active site pocket. To probe the molecular basis of this feature, we performed detailed thermodynamic studies of the binding of structurally similar ligands, which differed with respect to the "cap", "linker", and "metal-binding" regions of the suberoylanilide hydroxamic acid (SAHA) pharmacophore, to HDAC8. The experimental data revealed that although the enthalpic (ΔH°) and entropic (ΔS°) changes for the binding of individual SAHA analogues to HDAC8 were substantially different, their binding free energies (ΔG°) were markedly similar, conforming to a strong enthalpy-entropy compensation effect. This effect was further observed in the temperature-dependent thermodynamics of binding of all SAHA analogues to the enzyme. Notably, in contrast to other metalloenzymes, our isothermal titration calorimetry experiments (performed in different buffers of varying ionization enthalpies) suggest that depending on the ligand, its zinc-binding group may or may not be deprotonated upon the binding to HDAC8. Furthermore, the heat capacity changes (ΔCp°) associated with the ligand binding to HDAC8 markedly differed from one SAHA analogue to the other, and such features could primarily be rationalized in light of the dynamic flexibility in the enzyme structure in conjunction with the reorganization of the active site resident water molecules. Arguments are presented that although the binding thermodynamic features described above would facilitate identification of weak to moderately tight-binding HDAC8 inhibitors (by a high-throughput and/or virtual screening of libraries of small molecules), they would pose major challenges for the structure-based rational design of highly potent and isozyme-selective inhibitors of human HDAC8.