The Incorporation of etanercept into a porous tri-layer scaffold for restoring and repairing cartilage tissue

Producción CientíficaCartilage diseases currently affect a high percentage of the world’s population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this could be controlled with biomaterials that act as an anti-inflammatory delivery system, capable of dosing these drugs over time in a specific area. The objective of this study was to incorporate etanercept (ETA) into porous three-layer scaffolds to decrease the inflammatory process in this soft tissue. ETA is a blocker of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). For this reason, the scaffold was built based on natural polymers, including chitosan and type I collagen. The scaffold was grafted next to subchondral bone using hydroxyapatite as filler. One of the biomaterials obtained was also crosslinked to compare its mechanical properties with the non-treated one. Both samples’ physicochemical properties were studied with SEM, microCT and photoacoustic imaging, and their rheological properties were also compared. The cell viability and proliferation of the human chondrocyte C28/I2 cell line were studied in vitro. An in vitro and in vivo controlled release study was evaluated in both specimens. The ETA anti-inflammatory effect was also studied by in vitro TNF-α and IL-6 production. The crosslinked and non-treated scaffolds had rheological properties suitable for this application. They were non-cytotoxic and favoured the in vitro growth of chondrocytes. The in vitro and in vivo ETA release showed desirable results for a drug delivery system. The TNF-α and IL-6 production assay showed that this drug was effective as an anti-inflammatory agent. In an in vivo OA mice model, safranin-O and fast green staining was carried out. The OA cartilage tissue improved when the scaffold with ETA was grafted in the damaged area. These results demonstrate that this type of biomaterial has high potential for clinical applications in tissue engineering and as a controlled drug delivery system in OA articular cartilage.European Union through the Erasmus PLUS doctoral fellowship (project 2015-1-NL01-KA 107-008639)VIDI personal grant (project 723.012.110

Multimodal imaging of hair follicle bulge-derived stem cells in a mouse model of traumatic brain injury

Traumatic brain injury (TBI) is a devastating event for which current therapies are limited. Stem cell transplantation may lead to recovery of function via different mechanisms, such as cell replacement through differentiation, stimulation of angiogenesis and support to the microenvironment. Adult hair follicle bulge-derived stem cells (HFBSCs) possess neuronal differentiation capacity, are easy to harvest and are relatively immune-privileged, which makes them potential candidates for autologous stem cell-based therapy. In this study, we apply in vivo multimodal, optical and magnetic resonance imaging techniques to investigate the behavior of mouse HFBSCs in a mouse model of TBI. HFBSCs expressed Luc2 and copGFP and were examined for their differentiation capacity in vitro. Subsequently, transduced HFBSCs, preloaded with ferumoxytol, were transplanted next to the TBI lesion (cortical region) in nude mice, 2 days after injury. Brains were fixed for immunohistochemistry 58 days after transplantation. Luc2- and copGFP-expressing, ferumoxytol-loaded HFBSCs showed adequate neuronal differentiation potential in vitro. Bioluminescence of the lesioned brain revealed survival of HFBSCs and magnetic resonance imaging identified their localization in the area of transplantation. Immunohistochemistry showed that transplanted cells stained for nestin and neurofilament protein (NF-Pan). Cells also expressed laminin and fibronectin but extracellular matrix masses were not detected. After 58 days, ferumoxytol could be detected in HFBSCs in brain tissue sections. These results show that HFBSCs are able to survive after brain transplantation and suggest that ce

Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

PURPOSE Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer. METHODS EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed. RESULTS Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers. CONCLUSION Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer

PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells

Ex vivo gene editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial ‘burst’ of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the γ-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9-PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases

Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants

Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair

Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts

Schomann T, Qunneis F, Widera D, Kaltschmidt C, Kaltschmidt B. Improved method for ex ovo-cultivation of developing chicken embryos for human stem cell xenografts. Stem Cells International. 2013;2013: 960958.The characterization of human stem cells for the usability in regenerative medicine is particularly based on investigations regarding their differentiation potential in vivo. In this regard, the chicken embryo model represents an ideal model organism. However, the access to the chicken embryo is only achievable by windowing the eggshell resulting in limited visibility and accessibility in subsequent experiments. On the contrary, ex ovo-culture systems avoid such negative side effects. Here, we present an improved ex ovo-cultivation method enabling the embryos to survive 13 days in vitro. Optimized cultivation of chicken embryos resulted in a normal development regarding their size and weight. Our ex ovo-approach closely resembles the development of chicken embryos in ovo, as demonstrated by properly developed nervous system, bones, and cartilage at expected time points. Finally, we investigated the usability of our method for trans-species transplantation of adult stem cells by injecting human neural crest-derived stem cells into late Hamburger and Hamilton stages (HH26–HH28/E5—E6) of ex ovo-incubated embryos. We demonstrated the integration of human cells allowing experimentally easy investigation of the differentiation potential in the proper developmental context. Taken together, this ex ovo-method supports the prolonged cultivation of properly developing chicken embryos enabling integration studies of xenografted mammalian stem cells at late developmental stages

Rare-Earth-Metal (Nd3+, Ce3+ and Gd3+)-Doped CaF2: Nanoparticles for Multimodal Imaging in Biomedical Applications

Here, we describe the synthesis of a novel type of rare-earth-doped nanoparticles (NPs) for multimodal imaging, by combining the rare-earth elements Ce, Gd and Nd in a crystalline host lattice consisting of CaF2 (CaF2: Ce, Gd, Nd). CaF2: Ce, Gd, Nd NPs are small (15–20 nm), of uniform shape and size distribution, and show good biocompatibility and low immunogenicity in vitro. In addition, CaF2: Ce, Gd, Nd NPs possess excellent optical properties. CaF2: Ce, Gd, Nd NPs produce downconversion emissions in the second near-infrared window (NIR-II, 1000–1700 nm) under 808 nm excitation, with a strong emission peak at 1056 nm. Excitation in the first near- infrared window (NIR-I, 700–900 nm) has the advantage of deeper tissue penetration power and reduced autofluorescence, compared to visible light. Thus, CaF2: Ce, Gd, Nd NPs are ideally suited for in vivo fluorescence imaging. In addition, the presence of Gd3+ makes the NPs intrinsically monitorable by magnetic resonance imaging (MRI). Moreover, next to fluorescence and MR imaging, our results show that CaF2: Ce, Gd, Nd NPs can be used as imaging probes for photoacoustic imaging (PAI) in vitro. Therefore, due to their biocompatibility and suitability as multimodal imaging probes, CaF2: Ce, Gd, Nd NPs exhibit great potential as a traceable imaging agent in biomedical applications

Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea

Stem-cell-based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe-to-profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non-invasive in vivo visualization of cells and long-term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co-expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase-expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427-440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists

Combination of photodynamic therapy and stimulator of interferon genes (STING) agonist inhibits colorectal tumor growth and recurrence

Cancer Signaling networks and Molecular Therapeutic

Lentiviral transduction and subsequent loading with nanoparticles do not affect cell viability and proliferation in hair-follicle-bulge-derived stem cells in vitro

The application of stem cells in the treatment of various degenerative diseases is highly promising. However, cell-based therapy could be limited by the problem of low viability of grafted cells and uncertainty about their fate. The combination of molecular imaging and contrast-enhanced MRI may give more insight into the survival and behavior of grafted stem cells. We explore hair-follicle-bulge-derived stem cells (HFBSCs) as a potential candidate for autologous cell-based therapy. HFBSCs are transduced with a lentiviral construct with genes coding for bioluminescent (Luc2) and fluorescent (copGFP) reporter proteins, and subsequently loaded with magnetic nanoparticles to enable MRI visualization. Thus, we investigate for the first time if lentiviral transduction and cellular loading with nanoparticles have a cytotoxic effect upon these stem cells. Transduction efficiency, proliferation rate, cell viability and reporter protein co-expression during long-term culture of transduced HFBSCs were studied using fluorescence and bioluminescence microscopy. In addition, the effect of TMSR50 nanoparticles on proliferation and viability was investigated using the MTS assay and bioluminescence microscopy. The amount of TMSR50-loaded HFBSCs needed to reach signal threshold for MRI was assessed using an agarose phantom. Transduction with the Luc2-copGFP construct did not influence senescence, proliferation, doubling time, and differentiation of the HFBSCs. CopGFP expression was visible immediately after transduction and persisted for at least 15 passages, concomitantly with Luc2 expression. Cellular loading with TMSR50 nanoparticles did not affect cell viability and proliferation. The results imply that combined MRI and bioluminescence imaging may enable in vivo localization and long-term monitoring of grafted viable HFBSCs. Copyright © 2016 John Wiley & Sons, Ltd