Search Results (74)

Surface functionalization of dental implant surfaces has been a developing field in biomaterial research. This study aimed to obtain self-assembled monolayers (SAMs) using carboxyethylphosphonic acid on the surface of titanium (Ti) screws, and assessed the surface characteristics, biomechanical, and cellular behavior on the obtained specimens. This study had three groups, i.e., a control (untreated screws), a test group treated with phosphonic acid, and a third group with treated acid and bone morphogenetic protein (BMP-2) for in vitro analysis of cell lines. The assessed parameters included surface wettability, surface characteristics using scanning electron microscopy (SEM), protein immobilization, and cellular behavior of fibroblasts and mesenchymal stem cells of adipose tissue (MSCat cells). For surface wettability, a Welch test was performed to compare the contact angles between control (67 ± 1.83) and test (18.84 ± 0.72) groups, and a difference was observed in the mean measurements, but was not statistically significant. The SEM analysis showed significant surface roughness on the test screws and the cellular behavior of fibroblasts, and MSCat cells were significantly improved in this group, with fibroblasts having a polygonal shape with numerous vesicles and MSCat cells stable and uniformly coating the test Ti surface. Surface biofunctionalization of Ti surfaces with phosphonic acid showed promising results in this study, but remains to be clinically validated for its applications. Full article

One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Genetically encoded fluorescent biosensors constitute a class of imaging agents that enable visualization of biological processes and events directly in situ, preserving the native biological context and providing detailed insight into their localization and dynamics in cells. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically encoded fluorescent biosensors in drug screening. This review summarizes results of the studies that have been conducted in the last years toward the fabrication of genetically encoded fluorescent biosensors for biomedical applications with a comprehensive discussion on the challenges, future trends, and potential inputs needed for improving them. Full article

We developed a new probe, Gd-DO3A-Am-PBA, for imaging tumors. Our results showed active targeting of Gd-DO3A-Am-PBA to sialic acid (SA) moieties, with increased cellular labeling in vitro and enhanced tumor accumulation and retention in vivo, compared to the commercial Gadovist. The effectiveness of our newly synthesized probe lies in its adequate retention phase, which is expected to provide a suitable time window for tumor diagnosis and a faster renal clearance, which will reduce toxicity risks when translated to clinics. Hence, this study can be extended to other tumor types that express SA on their surface. Targeting and MR imaging of any type of tumors can also be achieved by conjugating the newly synthesized contrast agent with specific antibodies. This study thus opens new avenues for drug delivery and tumor diagnosis via imaging. Full article

Polyphenols have been shown to possess several beneficial properties, including properties involved in the prevention or treatment of cancer. Among these polyphenols, a leading role is played by dihydroxyphenylethanol (DPE), the most powerful antioxidant compound contained in the olive oil. DPE has been previously reported to induce endoplasmic reticulum (ER) stress and to reduce cell survival in colon cancer, one of the most common and aggressive cancers in developed countries. In this study, we further investigated the activation of UPR by DPE and explored the roles of the three UPR sensors, inositol-requiring enzyme (IRE) 1 alpha, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor (ATF6), in the cell death–survival decision of wt and mutp53 colon cancer cells and the underlying mechanisms involved. We also unveiled a new interplay between ATF6 and wt, as well as mutp53, which may have important implications in cancer therapy. Full article

Hemophilia A is a bleeding disease caused by loss of coagulation factor VIII (FVIII) function. Although prophylactic FVIII infusion prevents abnormal bleeding, disability and joint damage in hemophilia patients are common. The cost of treatment is among the highest for a single disease, and the adverse effects of repeated infusion are still an issue that has not been addressed. In this study, we established a nonviral gene therapy strategy to treat FVIII knockout (FVIII KO) mice. A novel gene therapy approach was developed using dipalmitoylphosphatidylcholine formulated with iron oxide (DPPC-Fe₃O₄) to carry the B-domain-deleted (BDD)-FVIII plasmid, which was delivered into the FVIII KO mice via tail vein injection. Here, a liver-specific albumin promoter-driven BDD-FVIII plasmid was constructed, and the binding ability of circular DNA was confirmed to be more stable than that of linear DNA when combined with DPPC-Fe₃O₄ nanoparticles. The FVIII KO mice that received the DPPC-Fe₃O₄ plasmid complex were assessed by staining the ferric ion of DPPC-Fe₃O₄ nanoparticles with Prussian blue in liver tissue. The bleeding of the FVIII KO mice was improved in a few weeks, as shown by assessing the activated partial thromboplastin time (aPTT). Furthermore, no liver toxicity, thromboses, deaths, or persistent changes after nonviral gene therapy were found, as shown by serum liver indices and histopathology. The results suggest that this novel gene therapy can successfully improve hemostasis disorder in FVIII KO mice and might be a promising approach to treating hemophilia A patients in clinical settings. Full article

The purpose of this study was to investigate, in vitro and in vivo, the suitability of chitosan (CHS) scaffolds produced by the net-shape-nonwoven (NSN) technology, for use as bone graft substitutes in a critical-size femoral bone defect in rats. For in vitro investigations, scaffolds made of CHS, mineralized collagen (MCM), or human cancellous bone allograft (CBA) were seeded with human telomerase-immortalized mesenchymal stromal cells (hTERT-MSC), incubated for 14 days, and thereafter evaluated for proliferation and osteogenic differentiation. In vivo, CHS, MCM and CBA scaffolds were implanted into 5 mm critical-size femoral bone defects in rats. After 12 weeks, the volume of newly formed bone was determined by microcomputed tomography (µCT), while the degree of defect healing, as well as vascularization and the number of osteoblasts and osteoclasts, was evaluated histologically. In vitro, CHS scaffolds showed significantly higher osteogenic properties, whereas treatment with CHS, in vivo, led to a lower grade of bone-healing compared to CBA and MCM. While chitosan offers a completely new field of scaffold production by fibers, these scaffolds will have to be improved in the future, regarding mechanical stability and osteoconductivity. Full article

Uptake of particles by cells involves various natural mechanisms that are essential for their biological functions. The same mechanisms are used in the engulfment of synthetic colloidal drug carriers, while the extent of the uptake affects the biological performance and selectivity. Thus far, little is known regarding the effect of external biomechanical stimuli on the capacity of the cells to uptake nano and micro carriers. This is relevant for anchorage-dependent cells that have detached from surfaces or for cells that travel in the body such as tumor cells, immune cells and various circulating stem cells. In this study, we hypothesize that cellular deformability is a crucial physical effector for the successful execution of the phagocytosis-like uptake in cancer cells. To test this assumption, we develop a well-controlled tunable method to compare the uptake of inert particles by cancer cells in adherent and non-adherent conditions. We introduce a self-designed 3D-printed apparatus, which enables constant stirring while facilitating a floating environment for cell incubation. We reveal a mechanically mediated phagocytosis-like behavior in various cancer cells, that was dramatically enhance in the detached cell state. Our findings emphasize the importance of including proper biomechanical cues to reliably mimic certain physiological scenarios. Beyond that, we offer a cost-effective accessible research tool to study mixed cultures for both adherent and non-adherent cells. Full article

The tissue engineering approach in osteoarthritic cell therapy often requires the delivery of a substantially high cell number due to the low engraftment efficiency as a result of low affinity binding of implanted cells to the targeted tissue. A modification towards the cell membrane that provides specific epitope for antibody binding to a target tissue may be a plausible solution to increase engraftment. In this study, we intercalated palmitated protein G (PPG) with mesenchymal stem cells (MSCs) and antibody, and evaluated their effects on the properties of MSCs either in monolayer state or in a 3D culture state (gelatin microsphere, GM). Bone marrow MSCs were intercalated with PPG (PPG-MSCs), followed by coating with type II collagen antibody (PPG-MSC-Ab). The effect of PPG and antibody conjugation on the MSC proliferation and multilineage differentiation capabilities both in monolayer and GM cultures was evaluated. PPG did not affect MSC proliferation and differentiation either in monolayer or 3D culture. The PPG-MSCs were successfully conjugated with the type II collagen antibody. Both PPG-MSCs with and without antibody conjugation did not alter MSC proliferation, stemness, and the collagen, aggrecan, and sGAG expression profiles. Assessment of the osteochondral defect explant revealed that the PPG-MSC-Ab micromass was able to attach within 48 h onto the osteochondral surface. Antibody-conjugated MSCs in GM culture is a potential method for targeted delivery of MSCs in future therapy of cartilage defects and osteoarthritis. Full article

Matrix-assisted autologous chondrocyte implantation (MACI) has shown promising results for cartilage repair, combining cultured chondrocytes and hydrogels, including alginate. The ability of chondrocytes for MACI is limited by different factors including donor site morbidity, dedifferentiation, limited lifespan or poor proliferation in vitro. Mesenchymal stem cells could represent an alternative for cartilage regeneration. In this study, we propose a MACI scaffold consisting of a mixed alginate-agarose hydrogel in combination with human dental pulp stem cells (hDPSCs), suitable for cartilage regeneration. Scaffolds were characterized according to their rheological properties, and their histomorphometric and molecular biology results. Agarose significantly improved the biomechanical behavior of the alginate scaffolds. Large scaffolds were manufactured, and a homogeneous distribution of cells was observed within them. Although primary chondrocytes showed a greater capacity for chondrogenic differentiation, hDPSCs cultured in the scaffolds formed large aggregates of cells, acquired a rounded morphology and expressed high amounts of type II collagen and aggrecan. Cells cultured in the scaffolds expressed not only chondral matrix-related genes, but also remodeling proteins and chondrocyte differentiation factors. The degree of differentiation of cells was proportional to the number and size of the cell aggregates that were formed in the hydrogels. Full article

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix. Full article

Because cartilage has limited regenerative capability, a fully efficient advanced therapy medicinal product is needed to treat severe cartilage damage. We evaluated a novel biomaterial obtained by decellularizing sturgeon chondral endoskeleton tissue for use in cartilage tissue engineering. In silico analysis suggested high homology between human and sturgeon collagen proteins, and ultra-performance liquid chromatography confirmed that both types of cartilage consisted mainly of the same amino acids. Decellularized sturgeon cartilage was recellularized with human chondrocytes and four types of human mesenchymal stem cells (MSC) and their suitability for generating a cartilage substitute was assessed ex vivo and in vivo. The results supported the biocompatibility of the novel scaffold, as well as its ability to sustain cell adhesion, proliferation and differentiation. In vivo assays showed that the MSC cells in grafted cartilage disks were biosynthetically active and able to remodel the extracellular matrix of cartilage substitutes, with the production of type II collagen and other relevant components, especially when adipose tissue MSC were used. In addition, these cartilage substitutes triggered a pro-regenerative reaction mediated by CD206-positive M2 macrophages. These preliminary results warrant further research to characterize in greater detail the potential clinical translation of these novel cartilage substitutes. Full article

The accumulation of extracellular β-amyloid (Aβ) plaques within the brain is unique to Alzheimer’s disease (AD) and thought to induce synaptic deficits and neuronal loss. Optimal therapies should tackle the core AD pathophysiology and prevent the decline in memory and cognitive functions. This study aimed to evaluate the therapeutic performance of mesenchymal stem cell-derived exosomes (MSC-exosomes), which are secreted membranous elements encapsulating a variety of MSC factors, on AD. A human neural cell culture model with familial AD (FAD) mutations was established and co-cultured with purified MSC-exosomes. 2-[¹⁸F]Fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) and novel object recognition (NOR) testing were performed before/after treatment to evaluate the therapeutic effect in vivo. The AD-related pathology and the expression of neuronal memory/synaptic plasticity-related genes were also evaluated. The results showed that MSC-exosomes reduced Aβ expression and restored the expression of neuronal memory/synaptic plasticity-related genes in the cell model. [¹⁸F]FDG-PET imaging and cognitive assessment revealed a significant improvement in brain glucose metabolism and cognitive function in AD transgenic mice. The phase of neurons and astrocytes in the brain of AD mice were also found to be regulated after treatment with MSC-exosomes. Our study demonstrates the therapeutic mechanism of MSC-exosomes and provides an alternative therapeutic strategy based on cell-free MSC-exosomes for the treatment of AD. Full article

Extracellular vesicles (EVs) are membrane-enclosed structures ranging in size from about 60 to 800 nm that are released by the cells into the extracellular space; they have attracted interest as easily available biomarkers for cancer diagnostics. In this study, EVs from plasma of control and prostate cancer patients were fractionated by differential centrifugation at 5000× g, 12,000× g and 120,000× g. The remaining supernatants were purified by ultrafiltration to produce EV-depleted free-circulating (fc) fractions. Spontaneous Raman and surface-enhanced Raman spectroscopy (SERS) at 785 nm excitation using silver nanoparticles (AgNPs) were employed as label-free techniques to collect fingerprint spectra and identify the fractions that best discriminate between control and cancer patients. SERS spectra from 10 µL droplets showed an enhanced Raman signature of EV-enriched fractions that were much more intense for cancer patients than controls. The Raman spectra of dehydrated pellets of EV-enriched fractions without AgNPs were dominated by spectral contributions of proteins and showed variations in S-S stretch, tryptophan and protein secondary structure bands between control and cancer fractions. We conclude that the AgNPs-mediated SERS effect strongly enhances Raman bands in EV-enriched fractions, and the fractions, EV12 and EV120 provide the best separation of cancer and control patients by Raman and SERS spectra. Full article