Chemical Properties of Human Dentin Blocks and Vertical Augmentation by Ultrasonically Demineralized Dentin Matrix Blocks on Scratched Skull without Periosteum of Adult-Aged Rats

Vertical augmentation is one of the most challenging techniques in bone engineering. Several parameters, such mechano-chemical characteristics, are important to optimize vertical bone regeneration using biomaterials. The aims of this study were to chemically characterize human dentin blocks (calcified demineralized dentin matrix: CDM, partially demineralized dentin matrix: PDDM and completely demineralized dentin matrix: CDDM) (2 × 2 × 1 mm3) chemically and evaluate the behavior of PDDM blocks on non-scratched or scratched skulls without periosteum of adult rats (10–12 months old, female) as a vertical augmentation model. The dissolved efficiency of CDM showed 32.3% after ultrasonic demineralization in 1.0 L of 2% HNO3 for 30 min. The 30 min-demineralized dentin was named PDDM. The SEM images of PDDM showed the opening of dentinal tubes, nano-microcracks and the smooth surface. In the collagenase digestion test, the weight-decreasing rates of CDM, PDDM and CDDM were 9.2%, 25.5% and 78.3% at 12 weeks, respectively. CDM inhibited the collagenase digestion, compared with PDDM and CDDM. In the PDDM onlay graft on an ultrasonically scratched skull, the bone marrow-space opening from original bone was found in the bony bridge formation between the human PDDM block and dense skull of adult senior rats at 4 and 8 weeks. On the other hand, in the cases of the marrow-space closing in both non-scratched skulls and scratched skulls, the bony bridge was not formed. The results indicated that the ultrasonic scratching into the compact parietal bone might contribute greatly to the marrow-space opening from skull and the supply of marrow cells, and then bony bridge formation could occur in the vertical augmentation model without a periosteum.

Modification of Human Pericardium by Chemical Crosslinking

Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.

Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

Background Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. Results We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. Conclusions The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.