Pulse Electrochemical Driven Rapid Layer-by-Layer Assembly of Polydopamine and Hydroxyapatite Nanofilms via Alternative Redox <i>in Situ</i> Synthesis for Bone Regeneration

Polydopamine (PDA) is an important candidate material for the surface modification of biomedical devices because of its good adhesiveness and biocompatibility. However, PDA nanofilms lack osteoinductivity, limiting their applications in bone tissue engineering. Hydroxyapatite nanoparticles (HA-NPs) are the major component of natural bone, which can be used to effectively enhance the osteoinductivity of PDA nanofilms. Herein, we developed a pulse electrochemical driven layer-by-layer (PED-LbL) assembly process to rapidly deposit HA-NPs and PDA (HA-PDA) multilayer nanofilms. In this process, PDA and HA-NPs are <i>in situ</i> synthesized in two sequential oxidative and reductive pulses in each electrochemical deposition cycle and alternately deposited on the substrate surfaces. PDA assists the <i>in situ</i> synthesis of HA-NPs by working as a template, which avoids the noncontrollable HA nucleation and aggregation. The HA-PDA multilayer nanofilms serve as a tunable reservoir to deliver bone morphogenetic protein-2 and exhibit high osteoinductivity both <i>in vitro</i> and <i>in vivo</i>. This PED-LbL assembly process breaks the limitation of traditional LbL assembly, allowing not only the rapid assembly of oppositely charged polyelectrolytes but also the <i>in situ</i> synthesis of organic/inorganic NPs that are uniformly incorporated in the nanofilm. It has broad applications in the preparation of versatile surface coatings on various biomedical devices

Mussel-Inspired Tissue-Adhesive Hydrogel Based on the Polydopamine–Chondroitin Sulfate Complex for Growth-Factor-Free Cartilage Regeneration

Glycosaminoglycan-based hydrogels are widely used for cartilage repair because glycosaminoglycans are the main component of the cartilage extracellular matrix and can maintain chondrocyte functions. However, most of the glycosaminoglycan-based hydrogels are negatively charged and cell-repellant, and they cannot host cells or favor tissue regeneration. Inspired by mussel chemistry, we designed a polydopamine–chondroitin sulfate–polyacrylamide (PDA–CS–PAM) hydrogel with tissue adhesiveness and super mechanical properties for growth-factor-free cartilage regeneration. Thanks to the abundant reactive catechol groups on the PDA, a cartilage-specific PDA–CS complex was formed by the self-assembly of PDA and CS, and then the PDA–CS complex was homogenously incorporated into an elastic hydrogel network. This catechol-group-enriched PDA–CS complex endowed the hydrogel with good cell affinity and tissue adhesiveness to facilitate cell adhesion and tissue integration. Compared with bare CS, the PDA–CS complex in the hydrogel was more effective in exerting its functions on adhered cells to upregulate chondrogenic differentiation. Because of the synergistic effects of noncovalent interactions caused by the PDA–CS complex and covalently cross-linked PAM network, the hydrogel exhibited super resilience and toughness, meeting the mechanical requirement of cartilage repair. Collectively, this tissue-adhesive and tough PDA–CS–PAM hydrogel with good cell affinity creates a growth-factor-free and biomimetic microenvironment for chondrocyte growth and cartilage regeneration and sheds light on the development of growth-factor-free biomaterials for cartilage repair

Supplementary Data from Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor–engineered T Cells

Supplementary methods and data</p

Supplementary Figure 1 from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure 1 - PDF file 53K, Antigen specific, HLA-restricted and dose-dependent recognition of the MM cell lines U266 and UM9, but not of accessory cells by CD8+ and CD4+ mHag-specific CTLs. The CD4+ (A) and CD8+ (B) CTLs were co-cultured with the luc+ MM cell lines UM9 and U266 in different effector to target ratios. The HLA- and mHag typing of these MM cell lines are indicated. MM cell survival was determined 24 h after addition of T cells by CS-BLI. Results represent the mean values of triplicate cultures (+/- SEM). Results are representative of 3 independent assays. In (C and D) CD4+ (C) and CD8+ (D) CTLs were co-cultured with accessory cells. Accessory cell survival was determined 24h after addition of T cells by FACS annexine V/ propidium iodide staining of CD4 and CD8 negative cells in the co-cultures. Results are depicted as % surviving cells, measured as relative number annexin V/propidium iodide negative cells, compared to no T cells control</p

Supplementary Figure 6 from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure 6 - PDF file 74K, Synergistic interaction of YM155 and CTLs. In A, the effect of YM155 on survivin and MCL-1 protein levels of MM cells is shown. The cells were cultured with YM155 during 24h and the western blot assays were carried out as indicated in material methods. Band intensities were quantified using image J software, calculated as relative values to the control protein tubuline and plotted as relative to the expression levels of cells cultured without YM155. In B, the type of interaction between YM155 and CTLs (see figure 5C-D) in the presence of accessory cells was analyzed using Compusyn software (version 1.0, 2004 �). In the plots (A) the CI values corresponding to the doses in figure 5 are shown. A CI value of 1 indicates synergy, additive effects or antagonism, respectively. In C the supernatants of assays as described in Fig 5C-D granzyme B release was measured using ELISA (B). Results are expressed as the mean values of the triplicate cultures. Error bars represent the SEM. Results are representative of 3 independent assays</p

Supplementary Figure 3 from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure 3 - PDF file 55K, RGDw reduces adhesion of MM cells to accessory cells. Adherent HS-5 en HUVEC accessory cells were incubated with RGDw or an irrelevant peptide for 1hour. After washing away the unbound peptide, UM9 (A) or U266 (B) cells were added and incubated overnight. Plates were reversely centrifuged to determine adhesion to accessory cells. Results represent the mean values of triplicate cultures (+/- SEM). Differences in adhesion were tested by unpaired two tailed student's t test. *= p<0.05; **= p<0.01, ***= p<0.001</p

Supplementary Figure Legends from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure Legends - PDF file 73K, Legends for supplementary figures</p

Supplementary Figure 4 from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure 4 - PDF file 219K, FasL upregulation on CTLs upon MM cell recognition is not hampered by presence of accessory cells. CD4+ (A) and CD8+ (B) CTLs were cultured alone (unstimulated) or in the presence of tumor cells and in presence or absence of accessory cells as indicated. After 3 hours of co-incubation CTLs were evaluated for CD178 (Fas ligand) expression. Percentage of FasL expressing cells is indicated</p

Supplementary Figure 5 from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure 5 - PDF file 41K, Bortezomib does not improve the CTL mediated lysis of Fas negative L363 cellsL3L3 reactive CD8+CTLs were incubated at the indicated effector to target ratios with luc-transduced L3L3 cells alone or in the presence of various dilutions of bortezomib. The CTL mediated and bortezomib mediated lysis of L3L3 cells was determined after 48 hours by BLI. Results are depicted as % surviving cells using the triplicate cultures without T cells and bortezomib as the 100% survival control</p

Supplementary Figure 2 from Accessory Cells of the Microenvironment Protect Multiple Myeloma from T-Cell Cytotoxicity through Cell Adhesion-Mediated Immune Resistance

Supplementary Figure 2 - PDF file 50K, Accessory cells protect MM cells from WT-1 specific CTLs. U266 cells were co-cultured with WT-1 specific T cells in presence and absence of different accessory cells. MM cell viability was determined 24h after addition of T cells by CS-BLI (A). Granzyme B production in the supernatant was determined by ELISA (B). Significance of inhibition of MM cell survival in the presence of accessory cells was tested by unpaired two tailed student's t test. *= p<0.05; **= p<0.01</p