A Broad Influenza Vaccine Based on a Heat-Activated, Tissue-Restricted Replication-Competent Herpesvirus.

Vaccination with transiently activated replication-competent controlled herpesviruses (RCCVs) expressing influenza A virus hemagglutinins broadly protects mice against lethal influenza virus challenges. The non-replicating RCCVs can be activated to transiently replicate with high efficiency. Activation involves a brief heat treatment to the epidermal administration site in the presence of a drug. The drug co-control is intended as a block to inadvertent reactivation in the nervous system and, secondarily, viremia under adverse conditions. While the broad protective effects observed raise an expectation that RCCVs may be developed as universal flu vaccines, the need for administering a co-activating drug may dampen enthusiasm for such a development. To replace the drug co-control, we isolated keratin gene promoters that were active in skin cells but inactive in nerve cells and other cells in vitro. In a mouse model of lethal central nervous system (CNS) infection, the administration of a recombinant that had the promoter of the infected cell protein 8 (ICP8) gene of a wild-type herpes simplex virus 1 (HSV-1) strain replaced by a keratin promoter did not result in any clinical signs, even at doses of 500 times wild-type virus LD50. Replication of the recombinant was undetectable in brain homogenates. Second-generation RCCVs expressing a subtype H1 hemagglutinin (HA) were generated in which the infected cell protein 4 (ICP4) genes were controlled by a heat switch and the ICP8 gene by the keratin promoter. In mice, these RCCVs replicated efficiently and in a heat-controlled fashion in the epidermal administration site. Immunization with the activated RCCVs induced robust neutralizing antibody responses against influenza viruses and protected against heterologous and cross-group influenza virus challenges.

Very Broadly Effective Hemagglutinin-Directed Influenza Vaccines with Anti-Herpetic Activity.

A universal vaccine that generally prevents influenza virus infection and/or illness remains elusive. We have been exploring a novel approach to vaccination involving replication-competent controlled herpesviruses (RCCVs) that can be deliberately activated to replicate efficiently but only transiently in an administration site in the skin of a subject. The RCCVs are derived from a virulent wild-type herpesvirus strain that has been engineered to contain a heat shock promoter-based gene switch that controls the expression of, typically, two replication-essential viral genes. Additional safety against inadvertent replication is provided by an appropriate secondary mechanism. Our first-generation RCCVs can be activated at the administration site by a mild local heat treatment in the presence of an antiprogestin. Here, we report that epidermal vaccination with such RCCVs expressing a hemagglutinin or neuraminidase of an H1N1 influenza virus strain protected mice against lethal challenges by H1N1 virus strains representing 75 years of evolution. Moreover, immunization with an RCCV expressing a subtype H1 hemagglutinin afforded full protection against a lethal challenge by an H3N2 influenza strain, and an RCCV expressing a subtype H3 hemagglutinin protected against a lethal challenge by an H1N1 strain. Vaccinated animals continued to gain weight normally after the challenge. Protective effects were even observed in a lethal influenza B virus challenge. The RCCV-based vaccines induced robust titers of in-group, cross-group and even cross-type neutralizing antibodies. Passive immunization suggested that observed vaccine effects were at least partially antibody-mediated. In summary, RCCVs expressing a hemagglutinin induce robust and very broad cross-protective immunity against influenza.

Disruption of Proteostasis by Natural Products and Synthetic Compounds That Induce Pervasive Unfolding of Proteins: Therapeutic Implications.

Exposure of many cancer cells, including multiple myeloma cells, to cytotoxic concentrations of natural products celastrol and withaferin A or synthetic compounds of the IHSF series resulted in denaturation of a luciferase reporter protein. Proteomic analysis of detergent-insoluble extract fractions from HeLa-derived cells revealed that withaferin A, IHSF058 and IHSF115 caused denaturation of 915, 722 and 991 of 5132 detected cellular proteins, respectively, of which 440 were targeted by all three compounds. Western blots showed that important fractions of these proteins, in some cases approaching half of total protein amounts, unfolded. Relatively indiscriminate covalent modification of target proteins was observed; 1178 different proteins were modified by IHSF058. Further illustrating the depth of the induced proteostasis crisis, only 13% of these proteins detectably aggregated, and 79% of the proteins that aggregated were not targets of covalent modification. Numerous proteostasis network components were modified and/or found in aggregates. Proteostasis disruption caused by the study compounds may be more profound than that mediated by proteasome inhibitors. The compounds act by a different mechanism that may be less susceptible to resistance development. Multiple myeloma cells were particularly sensitive to the compounds. Development of an additional proteostasis-disrupting therapy of multiple myeloma is suggested.

Remote control of transgene expression using noninvasive near-infrared irradiation.

This study investigated whether noninvasive near-infrared (NIR) energy could be transduced into heat in deep-seated organs in which adenovirus type-5 vectors tend to accumulate, thereby activating heat shock protein (HSP) promoter-mediated transgene expression, without local administration of photothermal agents. NIR irradiation of the subdiaphragmatic and left dorsocranial part of the abdominal cavity of adult immunocompetent C3H/HeNRj mice with an 808-nm laser effectively increased the temperature of the irradiated regions of the liver and spleen, respectively, resulting in the accumulation of the heat-inducible HSP70 protein. Spatial control of transgene expression was achieved in the NIR-irradiated regions of the mice administered an adenoviral vector carrying a firefly luciferase (fLuc) coding sequence controlled by a human HSP70B promoter, as assessed by bioluminescence and immunohistochemistry analyses. Levels of reporter gene expression were modulated by controlling NIR power density. Spatial control of transgene expression through NIR-focused activation of the HSP70B promoter, as well as temporal regulation by administering rapamycin was achieved in the spleens of mice inoculated with an adenoviral vector encoding a rapamycin-dependent transactivator driven by the HSP70B promoter and an adenoviral vector carrying a fLuc coding sequence controlled by the rapamycin-activated transactivator. Mice that were administered rapamycin and exposed to NIR light expressed fLuc activity in the splenic region, whereas no activity was detected in mice that were only administered rapamycin or vehicle or only NIR-irradiated. Thus, in the absence of any exogenously supplied photothermal material, remote control of heat-induced transgene expression can be achieved in the liver and spleen by means of noninvasive NIR irradiation.

Withaferin A and Celastrol Overwhelm Proteostasis.

Withaferin A (WA) and celastrol (CEL) are major bioactive components of plants that have been widely employed in traditional medicine. The pleiotropic activities of plant preparations and the isolated compounds in vitro and in vivo have been documented in hundreds of studies. Both WA and CEL were shown to have anticancer activity. Although WA and CEL belong to different chemical classes, our synthesis of the available information suggests that the compounds share basic mechanisms of action. Both WA and CEL bind covalently to numerous proteins, causing the partial unfolding of some of these proteins and of many bystander proteins. The resulting proteotoxic stress, when excessive, leads to cell death. Both WA and CEL trigger the activation of the unfolded protein response (UPR) which, if the proteotoxic stress persists, results in apoptosis mediated by the PERK/eIF-2/ATF4/CHOP pathway or another UPR-dependent pathway. Other mechanisms of cell death may play contributory or even dominant roles depending on cell type. As shown in a proteomic study with WA, the compounds appear to function largely as electrophilic reactants, indiscriminately modifying reachable nucleophilic amino acid side chains of proteins. However, a remarkable degree of target specificity is imparted by the cellular context.

A Narrative Review of Cell-Based Approaches for Cranial Bone Regeneration.

Current cranial repair techniques combine the use of autologous bone grafts and biomaterials. In addition to their association with harvesting morbidity, autografts are often limited by insufficient quantity of bone stock. Biomaterials lead to better outcomes, but their effectiveness is often compromised by the unpredictable lack of integration and structural failure. Bone tissue engineering offers the promising alternative of generating constructs composed of instructive biomaterials including cells or cell-secreted products, which could enhance the outcome of reconstructive treatments. This review focuses on cell-based approaches with potential to regenerate calvarial bone defects, including human studies and preclinical research. Further, we discuss strategies to deliver extracellular matrix, conditioned media and extracellular vesicles derived from cell cultures. Recent advances in 3D printing and bioprinting techniques that appear to be promising for cranial reconstruction are also discussed. Finally, we review cell-based gene therapy approaches, covering both unregulated and regulated gene switches that can create spatiotemporal patterns of transgenic therapeutic molecules. In summary, this review provides an overview of the current developments in cell-based strategies with potential to enhance the surgical armamentarium for regenerating cranial vault defects.

Polylactide, Processed by a Foaming Method Using Compressed Freon R134a, for Tissue Engineering.

Fabricating polymeric scaffolds using cost-effective manufacturing processes is still challenging. Gas foaming techniques using supercritical carbon dioxide (scCO2) have attracted attention for producing synthetic polymer matrices; however, the high-pressure requirements are often a technological barrier for its widespread use. Compressed 1,1,1,2-tetrafluoroethane, known as Freon R134a, offers advantages over CO2 in manufacturing processes in terms of lower pressure and temperature conditions and the use of low-cost equipment. Here, we report for the first time the use of Freon R134a for generating porous polymer matrices, specifically polylactide (PLA). PLA scaffolds processed with Freon R134a exhibited larger pore sizes, and total porosity, and appropriate mechanical properties compared with those achieved by scCO2 processing. PLGA scaffolds processed with Freon R134a were highly porous and showed a relatively fragile structure. Human mesenchymal stem cells (MSCs) attached to PLA scaffolds processed with Freon R134a, and their metabolic activity increased during culturing. In addition, MSCs displayed spread morphology on the PLA scaffolds processed with Freon R134a, with a well-organized actin cytoskeleton and a dense matrix of fibronectin fibrils. Functionalization of Freon R134a-processed PLA scaffolds with protein nanoparticles, used as bioactive factors, enhanced the scaffolds' cytocompatibility. These findings indicate that gas foaming using compressed Freon R134a could represent a cost-effective and environmentally friendly fabrication technology to produce polymeric scaffolds for tissue engineering approaches.

Vitamin B9 derivatives as carriers of bioactive cations for musculoskeletal regeneration applications: Synthesis, characterization and biological evaluation.

The development of new drugs for musculoskeletal regeneration purposes has attracted much attention in the last decades. In this work, we present three novel vitamin B9 (folic acid)-derivatives bearing divalent cations (ZnFO, MgFO and MnFO), providing their synthesis mechanism and physicochemical characterization. In addition, a strong emphasis has been placed on evaluating their biological properties (along with our previously reported SrFO) using human mesenchymal stem cells (hMSC). In all the cases, pure folate derivatives (MFOs) with a bidentate coordination mode between the metal and the folate anion, and a 1:1 stoichiometry, were obtained in high yields. A non-cytotoxic dose of all the MFOs (50 µg/mL) was demonstrated to modulate by their own the mRNA profiles towards osteogenic-like or fibrocartilaginous-like phenotypes in basal conditions. Moreover, ZnFO increased the alkaline phosphatase activity in basal conditions, while both ZnFO and MnFO increased the matrix mineralization degree in osteoinductive conditions. Thus, we have demonstrated the bioactivity of these novel compounds and the suitability to further studied them in vivo for musculoskeletal regeneration applications.

Wear of hip prostheses increases serum IGFBP-1 levels in patients with aseptic loosening.

The biological mechanisms involved in aseptic loosening include inflammation-associated and bone resorption-associated processes. Coordinated cellular actions result in biochemical imbalances with devastating consequences for the joint. Given that this condition is not known for showing systemic signs, we investigated whether circulating levels of inflammation-related proteins are altered in patients with aseptic loosening. Our study included 37 patients who underwent revision surgery due to hip osteolysis and aseptic loosening and 31 patients who underwent primary total hip arthroplasty. Using antibody arrays, we evaluated the serum levels of 320 proteins in four patients from each group. The results showed differences in insulin-like growth factor-binding protein 1 (IGFBP-1) concentrations, which we then quantified using enzyme-linked immunosorbent assay tests in all study patients. The results confirmed that serum IGFBP-1 concentrations were higher in the revision surgery patients than in the hip arthroplasty patients. In vitro studies showed that exposure of human osteoblasts to titanium particles induced an IGFBP-1 release that further increased when exposure to particles was performed in media conditioned by human M1 macrophages. These findings suggest that elevated serum IGFBP-1 levels in patients with aseptic loosening can arise from increased local IGFBP-1 production in the inflammatory environment of the periprosthetic bed.

Chitosan-stabilized silver nanoclusters with luminescent, photothermal and antibacterial properties.

The aim of this paper is to achieve in situ photochemical synthesis of silver nanoclusters (AgNCs) stabilized by the multiple-amine groups of chitosan (Ch@AgNCs) with luminescent and photothermal properties. Ch@AgNCs were obtained by applying a fast and simple methodology previously described by our group. Direct functionalization of AgNCs with chitosan template provided new nanohybrids directly in water solution, both in the presence or absence of oxygen. The formation of hybrid AgNCs could be monitored by the rapid increase of the absorption and emission maximum band with light irradiation time. New Ch@AgNCs not only present photoluminescent properties but also photothermal properties when irradiated with near infrared light (NIR), transducing efficiently NIR into heat and increasing the temperature of the medium up to 23 °C. The chitosan polymeric shell associated to AgNCs works as a protective support stabilizing the metal cores, facilitating the storage of nanohybrids and preserving luminescent, photothermal and bactericide properties.

Herpes Simplex Viruses Whose Replication Can Be Deliberately Controlled as Candidate Vaccines.

Over the last few years, we have been evaluating a novel paradigm for immunization using viruses or virus-based vectors. Safety is provided not by attenuation or inactivation of vaccine viruses, but by the introduction into the viral genomes of genetic mechanisms that allow for stringent, deliberate spatial and temporal control of virus replication. The resulting replication-competent controlled viruses (RCCVs) can be activated to undergo one or, if desired, several rounds of efficient replication at the inoculation site, but are nonreplicating in the absence of activation. Extrapolating from observations that attenuated replicating viruses are better immunogens than replication-defective or inactivated viruses, it was hypothesized that RCCVs that replicate with wild-type-like efficiency when activated will be even better immunogens. The vigorous replication of the RCCVs should also render heterologous antigens expressed from them highly immunogenic. RCCVs for administration to skin sites or mucosal membranes were constructed using a virulent wild-type HSV-1 strain as the backbone. The recombinants are activated by a localized heat treatment to the inoculation site in the presence of a small-molecule regulator (SMR). Derivatives expressing influenza virus antigens were also prepared. Immunization/challenge experiments in mouse models revealed that the activated RCCVs induced far better protective immune responses against themselves as well as against the heterologous antigens they express than unactivated RCCVs or a replication-defective HSV-1 strain. Neutralizing antibody and proliferation responses mirrored these findings. We believe that the data obtained so far warrant further research to explore the possibility of developing effective RCCV-based vaccines directed to herpetic diseases and/or diseases caused by other pathogens.

Local delivery of bone morphogenetic protein-2 from near infrared-responsive hydrogels for bone tissue regeneration.

Achievement of spatiotemporal control of growth factors production remains a main goal in tissue engineering. In the present work, we combined inducible transgene expression and near infrared (NIR)-responsive hydrogels technologies to develop a therapeutic platform for bone regeneration. A heat-activated and dimerizer-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in hydrogels based on fibrin and plasmonic gold nanoparticles that transduced incident energy of an NIR laser into heat. In the presence of dimerizer, photoinduced mild hyperthermia induced the release of bioactive BMP-2 from NIR-responsive cell constructs. A critical size bone defect, created in calvaria of immunocompetent mice, was filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the heat-activated and dimerizer-dependent gene circuit. In animals that were treated with dimerizer, NIR irradiation of implants induced BMP-2 production in the bone lesion. Induction of NIR-responsive cell constructs conditionally expressing BMP-2 in bone defects resulted in the formation of new mineralized tissue, thus indicating the therapeutic potential of the technological platform.

Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells.

BACKGROUND: The mechanisms by which macrophage phenotype contributes to mesenchymal stem cells (MSC)-mediated bone repair remain unclear. In this work, we investigated the influence of factors released by human macrophages polarized to a pro-inflammatory or an anti-inflammatory phenotype on the ability of human MSC to attach, migrate, and differentiate toward the osteoblastic lineage. We focused on the role of TNF-α and IL-10, key pro-inflammatory and anti-inflammatory cytokines, respectively, in regulating MSC functions. METHODS: MSC were treated with media conditioned by pro-inflammatory or anti-inflammatory macrophages to study their influence in cell attachment, migration, and osteogenic differentiation. The involvement of TNF-α and IL-10 in the regulation of MSC functions was investigated using neutralizing antibodies and recombinant cytokines. RESULTS: Treatment of MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages promoted cell elongation and enhanced MSC ability to attach and migrate. These effects were more noticeable when MSC were treated with media from pro-inflammatory macrophages. Interestingly, MSC osteogenic activity was enhanced by factors released by anti-inflammatory macrophages, but not by pro-inflammatory macrophages. Significant IL-10 levels originated from anti-inflammatory macrophages enhanced MSC osteogenesis by increasing ALP activity and mineralization in MSC layers cultured under osteogenic conditions. Moreover, macrophage-derived IL-10 regulated the expression of the osteogenic markers RUNX2, COL1A1, and ALPL. Notably, low TNF-α levels secreted by anti-inflammatory macrophages increased ALP activity in differentiating MSC whereas high TNF-α levels produced by pro-inflammatory macrophages had no effects on osteogenesis. Experiments in which MSC were treated with cytokines revealed that IL-10 was more effective in promoting matrix maturation and mineralization than TNF-α. CONCLUSIONS: Factors secreted by pro-inflammatory macrophages substantially increased MSC attachment and migration whereas those released by anti-inflammatory macrophages enhanced MSC osteogenic activity as well as cell migration. IL-10 was identified as an important cytokine secreted by anti-inflammatory macrophages that potentiates MSC osteogenesis. Our findings provide novel insights into how environments provided by macrophages regulate MSC osteogenesis, which may be helpful to develop strategies to enhance bone regeneration.

Gold nanoparticles for the in situ polymerization of near-infrared responsive hydrogels based on fibrin.

Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-l-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Current technological development allows synthesis of gold nanoparticles (GNP) in a wide range of shapes and sizes, consistently and at scale. GNP, stable and easily functionalized, show low cytotoxicity and high biocompatibility. Allied to that, GNP present optoelectronic properties that have been exploited in a range of biomedical applications. Following a layer-by-layer functionalization approach, we prepared hollow GNP coated with a positively charged copolymer that enabled thrombin conjugation. The resulting nanomaterial efficiently catalyzed the formation of fibrin hydrogels which convert energy of the near infrared (NIR) into heat. The resulting NIR-responsive hydrogels can function as scaffolding for cells capable of controlled gene expression triggered by optical hyperthermia, thus allowing the deployment of therapeutic gene products in desired spatiotemporal frameworks.

Glycerylphytate compounds with tunable ion affinity and osteogenic properties.

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.

Immunoregulatory potential of mesenchymal stem cells following activation by macrophage-derived soluble factors.

BACKGROUND: Immunoregulatory capacity of mesenchymal stem cells (MSC) is triggered by the inflammatory environment, which changes during tissue repair. Macrophages are essential in mediating the inflammatory response after injury and can adopt a range of functional phenotypes, exhibiting pro-inflammatory and anti-inflammatory activities. An accurate characterization of MSC activation by the inflammatory milieu is needed for improving the efficacy of regenerative therapies. In this work, we investigated the immunomodulatory functions of MSC primed with factors secreted from macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype. We focused on the role of TNF-α and IL-10, prototypic pro-inflammatory and anti-inflammatory cytokines, respectively, as priming factors for MSC. METHODS: Secretion of immunoregulatory mediators from human MSC primed with media conditioned by human macrophages polarized toward a pro-inflammatory or an anti-inflammatory phenotype was determined. Immunomodulatory potential of primed MSC on polarized macrophages was studied using indirect co-cultures. Involvement of TNF-α and IL-10 in priming MSC and of PGE2 in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Collagen hydrogels were used to study MSC and macrophages interactions in a more physiological environment. RESULTS: Priming MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages enhanced their immunomodulatory potential through increased PGE2 secretion. We identified the pro-inflammatory cytokine TNF-α as a priming factor for MSC. Notably, the anti-inflammatory IL-10, mainly produced by pro-resolving macrophages, potentiated the priming effect of TNF-α. Collagen hydrogels acted as instructive microenvironments for MSC and macrophages functions and their crosstalk. Culturing macrophages on hydrogels stimulated anti-inflammatory versus pro-inflammatory cytokine secretion. Encapsulation of MSC within hydrogels increased PGE2 secretion and potentiated immunomodulation on macrophages, attenuating macrophage pro-inflammatory state and sustaining anti-inflammatory activation. Priming with inflammatory factors conferred to MSC loaded in hydrogels greater immunomodulatory potential, promoting anti-inflammatory activity of macrophages. CONCLUSIONS: Factors secreted by pro-inflammatory and anti-inflammatory macrophages activated the immunomodulatory potential of MSC. This was partially attributed to the priming effect of TNF-α and IL-10. Immunoregulatory functions of primed MSC were enhanced after encapsulation in hydrogels. These findings may provide insight into novel strategies to enhance MSC immunoregulatory potency.

Spatiotemporally-controlled transgene expression in hydroxyapatite-fibrin composite scaffolds using high intensity focused ultrasound.

Conventional tissue engineering approaches rely on scaffold-based delivery of exogenous proteins, genes, and/or cells to stimulate regeneration via growth factor signaling. However, scaffold-based approaches do not allow active control of dose, timing, or spatial localization of a delivered growth factor once the scaffold is implanted, yet these are all crucial parameters in promoting tissue regeneration. To address this limitation, we developed a stable cell line containing a heat-activated and rapamycin-dependent gene expression system. In this study, we investigate how high intensity focused ultrasound (HIFU) can spatiotemporally control firefly luciferase (fLuc) transgene activity both in vitro and in vivo by the tightly controlled generation of hyperthermia. Cells were incorporated into composite scaffolds containing fibrin and hydroxyapatite particles, which yielded significant increases in acoustic attenuation and heating in response to HIFU compared to fibrin alone. Using 2.5 MHz HIFU, transgene activation was observed at acoustic intensities of 201 W/cm2 and higher. Transgene activation was spatially patterned in the scaffolds by rastering HIFU at speeds up to 0.15 mm/s. In an in vivo study, a 67-fold increase in fLuc activity was observed in scaffolds exposed to HIFU and rapamycin versus rapamycin only at 2 days post implantation. Repeated activation of transgene expression was also demonstrated 8 days after implantation. No differences in in vivo scaffold degradation or compaction were observed between +HIFU and -HIFU groups. These results highlight the potential utility of using this heat-activated and rapamycin-dependent gene expression system in combination with HIFU for the controlled stimulation of tissue regeneration.

Pro-angiogenic near infrared-responsive hydrogels for deliberate transgene expression.

CuS nanoparticles (CuSNP) are degradable, readily prepared, inexpensive to produce and efficiently cleared from the body. In this work, we explored the feasibility of CuSNP to function as degradable near infrared (NIR) nanotransducers within fibrin-based cellular scaffolds. To prepare NIR-responsive CuSNP hydrogels, fibrinogen was dissolved in cell culture medium and supplemented with aqueous dispersions of CuSNP. Fibrinogen polymerization was catalyzed by the addition of thrombin. In some experiments, HUVEC, C3H/10T1/2 or C3H/10T1/2-fLuc cells, that harbor a heat-activated and rapamycin-dependent gene switch for regulating the expression of firefly luciferase transgene, were incorporated to the sol phase of the hydrogel. For in vivo experiments, hydrogels were injected subcutaneously in the back of adult C3H/HeN mice. Upon NIR irradiation, CuSNP hydrogels allowed heat-inducible and rapamycin-dependent transgene expression in cells contained therein, in vitro and in vivo. C3H/10T1/2 cells cultured in CuSNP hydrogels increased metabolic activity, survival rate and fibrinolytic activity, which correlated with changes at the transcriptome level. Media conditioned by CuSNP hydrogels increased viability of HUVEC which formed pseudocapillary structures and remodeled protein matrix when entrapped within these hydrogels. After long-term implantation, the skin patches that covered the CuSNP hydrogels showed increased capillary density which was not detected in mice implanted with matrices lacking CuSNP. In summary, NIR-responsive scaffolds harboring CuSNP offer compelling features in the tissue engineering field, as degradable implants with enhanced integration capacity in host tissues that can provide remote controlled deployment of therapeutic gene products. STATEMENT OF SIGNIFICANCE: Hydrogels composed of fibrin embedding copper sulfide nanoparticles (CuSNP) efficiently convert incident near infrared (NIR) energy into heat and can function as cellular scaffolding. NIR laser irradiation of CuSNP hydrogels can be employed to remotely induce spatiotemporal patterns of transgene expression in genetically engineered multipotent stem cells. CuSNP incorporation in hydrogel architecture accelerates the cell-mediated degradation of the fibrin matrix and induces pro-angiogenic responses that may facilitate the integration of these NIR-responsive scaffolds in host tissues. CuSNP hydrogels that harbor cells capable of controlled expression of therapeutic gene products may be well suited for tissue engineering as they are biodegradable, enhance implant vascularization and can be used to deploy growth factors in a desired spatiotemporal fashion.

Publisher Correction: Paracrine interactions between mesenchymal stem cells and macrophages are regulated by 1,25-dihydroxyvitamin D3.

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