PDGF-D Is Dispensable for the Development and Progression of Murine Alport Syndrome.

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. In vitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3-/- mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3-/- mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3-/- mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3-/-Pdgfd-/- mice compared with Col4a3-/-Pdgfd+/+ littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3-/- mice might explain the lack of effects in vivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.

The Role of Platelet-Derived Growth Factor in Focal Segmental Glomerulosclerosis.

BACKGROUND: FSGS is the final common pathway to nephron loss in most forms of severe or progressive glomerular injury. Although podocyte injury initiates FSGS, parietal epithelial cells (PECs) are the main effectors. Because PDGF takes part in fibrotic processes, we hypothesized that the ligand PDGF-B and its receptor PDGFR- ß participate in the origin and progression of FSGS. METHODS: We challenged Thy1.1 transgenic mice, which express Thy1.1 in the podocytes, with anti-Thy1.1 antibody to study the progression of FSGS. We investigated the role of PDGF in FSGS using challenged Thy1.1 mice, 5/6 nephrectomized mice, Col4 -/- (Alport) mice, patient kidney biopsies, and primary murine PECs, and challenged Thy1.1 mice treated with neutralizing anti-PDGF-B antibody therapy. RESULTS: The unchallenged Thy1.1 mice developed only mild spontaneous FSGS, whereas challenged mice developed progressive FSGS accompanied by a decline in kidney function. PEC activation, proliferation, and profibrotic phenotypic switch drove the FSGS. During disease, PDGF-B was upregulated in podocytes, whereas PDGFR- ß was upregulated in PECs from both mice and patients with FSGS. Short- and long-term treatment with PDGF-B neutralizing antibody improved kidney function and reduced FSGS, PEC proliferation, and profibrotic activation. In vitro , stimulation of primary murine PECs with PDGF-B recapitulated in vivo findings with PEC activation and proliferation, which was inhibited by PDGF-B antibody or imatinib. CONCLUSION: PDGF-B-PDGFR- ß molecular crosstalk between podocytes and PECs drives glomerulosclerosis and the progression of FSGS. PODCAST: This article contains a podcast at.

A Biodegradable Tissue Adhesive for Post-Extraction Alveolar Bone Regeneration under Ongoing Anticoagulation-A Microstructural Volumetric Analysis in a Rodent Model.

In addition to post-extraction bleeding, pronounced alveolar bone resorption is a very common complication after tooth extraction in patients undergoing anticoagulation therapy. The novel, biodegenerative, polyurethane adhesive VIVO has shown a positive effect on soft tissue regeneration and hemostasis. However, the regenerative potential of VIVO in terms of bone regeneration has not yet been explored. The present rodent study compared the post-extraction bone healing of a collagen sponge (COSP) and VIVO in the context of ongoing anticoagulation therapy. According to a split-mouth design, a total of 178 extraction sockets were generated under rivaroxaban treatment, of which 89 extraction sockets were treated with VIVO and 89 with COSP. Post-extraction bone analysis was conducted via in vivo micro-computed tomography (µCT), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX) after 5, 10, and 90 days. During the observation time of 90 days, µCT analysis revealed that VIVO and COSP led to significant increases in both bone volume and bone density (p ≤ 0.001). SEM images of the extraction sockets treated with either VIVO or COSP showed bone regeneration in the form of lamellar bone mass. Ratios of Ca/C and Ca/P observed via EDX indicated newly formed bone matrixes in both treatments after 90 days. There were no statistical differences between treatment with VIVO or COSP. The hemostatic agents VIVO and COSP were both able to prevent pronounced bone loss, and both demonstrated a strong positive influence on the bone regeneration of the alveolar ridge post-extraction.

Simple plant-based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor-targeted drug delivery and bioimaging in cancer therapy.

Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and the absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents, and other cargos. However, recombinant ferritin currently costs ~3.5 million € g-1 , presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5'-untranslated regions in PCPs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by an ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg-1 fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo.

Effect of Radical Polymerization Method on Pharmaceutical Properties of Π Electron-Stabilized HPMA-Based Polymeric Micelles.

Polymeric micelles are among the most extensively used drug delivery systems. Key properties of micelles, such as size, size distribution, drug loading, and drug release kinetics, are crucial for proper therapeutic performance. Whether polymers from more controlled polymerization methods produce micelles with more favorable properties remains elusive. To address this question, we synthesized methoxy poly(ethylene glycol)-b-(N-(2-benzoyloxypropyl)methacrylamide) (mPEG-b-p(HPMAm-Bz)) block copolymers of three different comparable molecular weights (∼9, 13, and 20 kDa), via both conventional free radical (FR) and reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers were subsequently employed to prepare empty and paclitaxel-loaded micelles. While FR polymers had relatively high dispersities (D ∼ 1.5-1.7) compared to their RAFT counterparts (D ∼ 1.1-1.3), they formed micelles with similar pharmaceutical properties (e.g., size, size distribution, critical micelle concentration, cytotoxicity, and drug loading and retention). Our findings suggest that pharmaceutical properties of mPEG-b-p(HPMAm-Bz) micelles do not depend on the synthesis route of their constituent polymers.

Inhibition of the glucocorticoid receptor attenuates proteinuric kidney diseases in multiple species.

BACKGROUND AND HYPOTHESIS: Glucocorticoids are the treatment of choice for proteinuric patients with minimal-change disease (MCD) and primary focal and segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes. METHODS: We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone. RESULTS: Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin-aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids, were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria. CONCLUSIONS: Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While, the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.

In vitro comparison of the osteogenic capability of human pulp stem cells on alloplastic, allogeneic, and xenogeneic bone scaffolds.

BACKGROUND: A rigorous search for alternatives to autogenous bone grafts to avoid invasiveness at the donor site in the treatment of maxillomandibular bone defects. Researchers have used alloplastic, allogeneic, and xenogeneic bone graft substitutes in clinical studies with varying degrees of success, although their in vitro effects on stem cells remain unclear. Dental pulp stem cells (DPSCs) can potentially enhance the bone regeneration of bone graft substitutes. The present in vitro study investigates the osteogenic capability of DPSCs on alloplastic (biphasic calcium phosphate [BCP]), allogeneic (freeze-dried bone allografts [FDBAs]), and xenogeneic (deproteinized bovine bone mineral [DBBM]) bone grafts. METHODS: Human DPSCs were seeded on 0.5 mg/ml, 1 mg/ml, and 2 mg/ml of BCP, FDBA, and DBBM to evaluate the optimal cell growth and cytotoxicity. Scaffolds and cell morphologies were analyzed by scanning electron microscopy (SEM). Calcein AM and cytoskeleton staining were performed to determine cell attachment and proliferation. Alkaline phosphatase (ALP) and osteogenesis-related genes expressions was used to investigate initial osteogenic differentiation. RESULTS: Cytotoxicity assays showed that most viable DPSCs were present at a scaffold concentration of 0.5 mg/ml. The DPSCs on the DBBM scaffold demonstrated a significantly higher proliferation rate of 214.25 ± 16.17 (p < 0.001) cells, enhancing ALP activity level and upregulating of osteogenesis-related genes compared with other two scaffolds. CONCLUSION: DBBP scaffold led to extremely high cell viability, but also promoted proliferation, attachment, and enhanced the osteogenic differentiation capacity of DPSCs, which hold great potential for bone regeneration treatment; however, further studies are necessary.

Theranostic Trigger-Responsive Carbon Monoxide-Generating Microbubbles.

Carbon monoxide (CO) is a gaseous signaling molecule that modulates inflammation, cell survival, and recovery after myocardial infarction. However, handling and dosing of CO as a compressed gas are difficult. Here, light-triggerable and magnetic resonance imaging (MRI)-detectable CO release from dimanganese decacarbonyl (CORM-1) are demonstrated, and the development of CORM-1-loaded polymeric microbubbles (COMB) is described as an ultrasound (US)- and MRI-imageable drug delivery platform for triggerable and targeted CO therapy. COMB are synthesized via a straightforward one-step loading protocol, present a narrow size distribution peaking at 2 µm, and show excellent performance as a CORM-1 carrier and US contrast agent. Light irradiation of COMB induces local production and release of CO, as well as enhanced longitudinal and transversal relaxation rates, enabling MRI monitoring of CO delivery. Proof-of-concept studies for COMB-enabled light-triggered CO release show saturation of hemoglobin with CO in human blood, anti-inflammatory differentiation of macrophages, reduction of hypoxia-induced reactive oxygen species (ROS) production, and inhibition of ischemia-induced apoptosis in endothelial cells and cardiomyocytes. These findings indicate that CO-generating MB are interesting theranostic tools for attenuating hypoxia-associated and ROS-mediated cell and tissue damage in cardiovascular disease.

Repetitive Treatment with Volatile Anesthetics Does Not Affect the In Vivo Plasma Concentration and Composition of Extracellular Vesicles in Rats.

BACKGROUND: Anesthetic-induced preconditioning (AIP) with volatile anesthetics is a well-known experimental technique to protect tissues from ischemic injury or oxidative stress. Additionally, plasmatic extracellular vesicle (EV) populations and their cargo are known to be affected by AIP in vitro, and to provide organ protective properties via their cargo. We investigated whether AIP would affect the generation of EVs in an in vivo rat model. METHODS: Twenty male Sprague Dawley rats received a repetitive treatment with either isoflurane or with sevoflurane for a duration of 4 or 8 weeks. EVs from blood plasma were characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM) and Western blot. A scratch assay (H9C2 cardiomyoblast cell line) was performed to investigate the protective capabilities of the isolated EVs. RESULTS: TEM images as well as Western blot analysis indicated that EVs were successfully isolated. The AIP changed the flotillin and CD63 expression on the EV surface, but not the EV concentration. The scratch assay did not show increased cell migration and/or proliferation after EV treatment. CONCLUSION: AIP in rats changed the cargo of EVs but had no effect on EV concentration or cell migration/proliferation. Future studies are needed to investigate the cargo on a miRNA level and to investigate the properties of these EVs in additional functional experiments.

A Hypercaloric Diet Induces Early Podocyte Damage in Aged, Non-Diabetic Rats.

BACKGROUND/AIMS: The number of patients of older age with metabolic syndrome, obesity, and associated kidney disease, which is characterized by podocyte damage, glomerular hypertrophy, and focal segmental glomerulosclerosis (FSGS), is increasing worldwide. Animal models that would reflect the development of such kidney diseases could facilitate the testing of drugs. We investigated the renal effects of a long-term high caloric diet in aged rats and the potential effects of drugs used to treat metabolic syndrome. METHODS: We analyzed nine-month-old male and female Sprague Dawley rats fed five months with a normal diet (control group) or high-fat-high-carbohydrate diet (HFHCD group). Two additional groups were fed with HFHCD and treated with drugs used in patients with metabolic syndrome, i.e., the glucagon-like peptide receptor 1 agonist liraglutide (HFHCD+liraglutide group) or metformin (HFHCD+metformin group). RESULTS: Except an increase of waist circumference as a sign of visceral obesity, the HFHCD diet did not induce metabolic syndrome or obesity. There were no significant changes in kidney function and all groups showed similar indices of glomerular injury, i.e., no differences in glomerular size or the number of glomeruli with FSGS or with FSGS-precursor lesions quantified by CD44 expression as a marker of parietal epithelial cell (PEC) activation. Analysis of ultrastructural morphology revealed mild podocyte stress and a decrease of glomerular nestin expression in the HFHCD group, whereas podocin and desmin were not altered. HFHCD did not promote fibrogenesis, however, treatment with liraglutide led to a slightly increased tubulointerstitial damage, immune cell infiltration, and collagen IV expression compared to the control and HFHCD groups. CONCLUSION: A five-month feeding with HFHCD in aged rats induced mild podocyte injury and microinflammation, which was not alleviated by liraglutide or metformin.

Functionalized Cellulose Nanocrystals for Cellular Labeling and Bioimaging.

Cellulose nanocrystals (CNCs) are unique and promising natural nanomaterials that can be extracted from native cellulose fibers by acid hydrolysis. In this study, we developed chemically modified CNC derivatives by covalent tethering of PEGylated biotin and perylenediimide (PDI)-based near-infrared organic dye and evaluated their suitability for labeling and imaging of different cell lines including J774A.1 macrophages, NIH-3T3 fibroblasts, HeLa adenocarcinoma cells, and primary murine dendritic cells. PDI-labeled CNCs showed a superior photostability compared to similar commercially available dyes under long periods of constant and high-intensity illumination. All CNC derivatives displayed excellent cytocompatibility toward all cell types and efficiently labeled cells in a dose-dependent manner. Moreover, CNCs were effectively internalized and localized in the cytoplasm around perinuclear areas. Thus, our findings demonstrate the suitability of these new CNC derivatives for labeling, imaging, and long-time tracking of a variety of cell lines and primary cells.

Implant removal using thermal necrosis-an in vitro pilot study.

OBJECTIVES: The purpose of this pilot porcine cadaver study was to evaluate the feasible temperature thresholds, which affect osteocyte viability and bone matrix in a preclinical setup, assessing the potential of thermal necrosis for implant removal for further in vivo investigations. MATERIALS AND METHODS: After implant bed preparation in the upper and lower jaw, temperature effects on the bone were determined, using two tempering pistons with integrated thermocouples. To evaluate threshold temperature and time intervals leading to bone necrosis, one piston generated warm temperatures at 49 to 56 °C for 10 s and the other generated cold temperatures at 5 to 1 °C for 30 s. Effects were assessed by a semi-quantitative, histomorphometrical scoring system, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). RESULTS: The bone matrix was significantly degenerated starting at 51 °C for 10 s and 5 °C for 30 s. The osteocyte condition indicated significant bone damage beginning at cold temperatures of 2 °C. Temperature inputs starting at 53 °C led to decalcification and swollen mitochondria, which lost the structure of their inner cristae. CONCLUSIONS: This study identified temperatures and durations, in both heat and cold, so that the number of samples may be kept low in further studies regarding temperature-induced bone necrosis. Levels of 51 °C for 10 s and 5 °C for 30 s have presented significant matrix degeneration. CLINICAL RELEVANCE: Temperature thresholds, potentially leading to thermo-explantation of dental implants and other osseointegrated devices, were identified.

In vivo and in vitro analysis in a rat model using zoledronate and alendronate medication: microbiological and scanning electron microscopy findings on peri-implant rat tissue.

BACKGROUND: The aim of the present study was to assess the development of bacterial deposits and morphological parameters around dental zirconia and titanium implants compared with natural teeth during systemic bisphosphonate medication. MATERIALS AND METHODS: Fifty-four rats were randomly allocated into one control group and two experimental groups (drug application of zoledronic and alendronic acid), with 18 animals in each group. After 4 weeks of drug delivery, either a zirconia or a titanium implant was immediately inserted. Microbiological analysis conducted 1 week, 8 weeks, and 12 weeks after surgery included total bacterial count and composition measurements. Samples were analyzed in a scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectroscopy (EDX). Bone cell morphology was analyzed by transmission electron microscopy (TEM). RESULTS: One week after surgery, titanium and zirconia implants of the alendronic acid and control group showed a significantly higher bacterial count when compared to natural teeth in rats with zoledronic acid administration (p < 0.01). Less significant differences were recorded after 3 months, at which time no inter-material differences were evaluated (p > 0.05). I n the control group, TEM analysis showed that the osteoblasts had a strongly developed endoplasmic reticulum. In contrast, the endoplasmic reticulum of the osteoblasts in drug-treated animals was significantly less developed, indicating less activity. CONCLUSIONS: Within the limits of this study, neither implant material was superior to the other at 3-month follow-up. With regard to the treatment and complications of patients with bisphosphonates, the implant material should not be an influencing factor. Bisphosphonates can be used in the rat model to reduce not only the activity of osteoclasts but also osteoblasts of the peri-implant bone.

Extracellular Vesicles-Loaded Fibrin Gel Supports Rapid Neovascularization for Dental Pulp Regeneration.

Rapid vascularization is required for the regeneration of dental pulp due to the spatially restricted tooth environment. Extracellular vesicles (EVs) released from mesenchymal stromal cells show potent proangiogenic effects. Since EVs suffer from rapid clearance and low accumulation in target tissues, an injectable delivery system capable of maintaining a therapeutic dose of EVs over a longer period would be desirable. We fabricated an EV-fibrin gel composite as an in situ forming delivery system. EVs were isolated from dental pulp stem cells (DPSCs). Their effects on cell proliferation and migration were monitored in monolayers and hydrogels. Thereafter, endothelial cells and DPSCs were co-cultured in EV-fibrin gels and angiogenesis as well as collagen deposition were analyzed by two-photon laser microscopy. Our results showed that EVs enhanced cell growth and migration in 2D and 3D cultures. EV-fibrin gels facilitated vascular-like structure formation in less than seven days by increasing the release of VEGF. The EV-fibrin gel promoted the deposition of collagen I, III, and IV, and readily induced apoptosis during the initial stage of angiogenesis. In conclusion, we confirmed that EVs from DPSCs can promote angiogenesis in an injectable hydrogel in vitro, offering a novel and minimally invasive strategy for regenerative endodontic therapy.

Biglycan evokes autophagy in macrophages via a novel CD44/Toll-like receptor 4 signaling axis in ischemia/reperfusion injury.

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44-/- mice but not Cd14-/- mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Transglutaminase enables highly hydrolytically and proteolytically stable crosslinking of collagen on titanium surfaces and promotes osteogenic differentiation of human mesenchymal stem cells.

Collagen with its bioactive ligand motives would be predestined as coating on bone implant surfaces like titanium hip stems to facilitate receptor-mediated cell adhesion and thereby improve early osseointegration. Unfortunately, collagen as coating exhibits very low proteolytic resistance in vivo. To overcome this limitation, different crosslinking methods of collagen (transglutaminase, GTA, EDC/NHS, riboflavin, and lysyl oxidase) with silanized titanium alloy (Ti6Al4V) were investigated in terms of degradation resistance, hydrolysis stability, tensile strength, and metabolic cell activity. The in vitro osteogenic differentiation ability of human mesenchymal stem cells (hMSCs) induced by the surface modification was evaluated by immunofluorescence of early osteogenic markers, Alizarin red staining, and energy dispersive X-ray spectroscopy. The expression of the adhesion-related protein vinculin was analyzed on the different functionalized surfaces. The results revealed that the enzymatic crosslinker transglutaminase offered high degradation resistance, tensile strength, and hydrolysis stability compared to the other crosslinking reagents tested. Remarkably, the adhesion sequences within the collagen were accessible to the hMSCs despite the transglutaminase crosslinking procedure. In conclusion, the organochemical functionalization of Ti6Al4V surfaces with collagen using transglutaminase holds great potential to facilitate an enhanced interaction with attached bone cells and thereby could potentially improve and accelerate osseointegration of a titanium-based bone implant in vivo.

Newly isolated Drexlerviridae phage LAPAZ is physically robust and fosters eradication of Klebsiella pneumoniae in combination with meropenem.

Due to the spread of multidrug resistance there is a renewed interest in using bacteriophages (briefly: phages) for controlling bacterial pathogens. The objective of this study was the characterization of a newly isolated phage (i.e. phage LAPAZ, vB_KpnD-LAPAZ), its antimicrobial activity against multidrug resistant Klebsiella pneumoniae and potential synergistic interactions with antibiotics. LAPAZ belongs to the family Drexlerviridae (genus: Webervirus) and lysed 30 % of tested strains, whereby four distinct capsular types can be infected. The genome consists of 51,689 bp and encodes 84 ORFs. The latent period is 30 min with an average burst size of 27 PFU/cell. Long-term storage experiments show that LAPAZ is significantly more stable in wastewater compared to laboratory media. A phage titre of 90 % persists up to 30 min at 50 ˚C and entire phage loss was seen only at temperatures > 66 ˚C. Besides stability against UV-C, antibacterial activity in liquid culture medium was consistent at pH values ranging from 4 to 10. Unlike exposure to phage or antibiotic alone, synergistic interactions and a complete bacterial eradication was achieved when combining LAPAZ with meropenem. In addition, synergism with the co-presence of ciprofloxacin was observed and phage resistance emergence could be delayed. Without co-addition of the antibiotic, phage resistant mutants readily emerged and showed a mixed pattern of drug sensitivity alterations. Around 88 % became less sensitive towards ceftazidime, meropenem and gentamicin. Conversely, around 44 % showed decreased resistance levels against ciprofloxacin. Whole genome analysis of a phage-resistant mutant with a 16-fold increased sensitivity towards ciprofloxacin revealed one de novo frameshift mutation leading to a gene fusion affecting two transport proteins belonging to the major facilitator-superfamily (MFS). Apparently, this mutation compromises ciprofloxacin efflux efficiency and further studies are warranted to understand how the non-mutated protein might be involved in phage-host adsorption.

Functionalization of a zirconia surface by covalently immobilized fibronectin and its effects on resistance to thermal, acid, and mechanical exposure.

Silane chemistry has emerged as a powerful tool for surface modification, offering a versatile means to enhance the properties of various substrates, such as dental implant abutment materials. In this study, we investigated the stability of the 3-aminopropyldiisopropylethoxysilane (APDS) layer on yttria-partially stabilized zirconia (Y-TZP) surfaces after mechanical, acid, and thermal treatment in order to simulate fluctuations within the oral cavity. To accomplish that, the viability of human gingival fibroblasts on APDS-modified surfaces after applied treatment strategies was assessed by live/dead staining. Moreover, the hydrolysis stability and enzymatic degradation resistance of crosslinked fibronectin to the APDS layer was examined by immunostaining and western blot. The results revealed that the applied modifications were not affected by the different treatment conditions and could withstand the fluctuations in the oral cavity. Furthermore, crosslinked fibronectin on silanized Y-TZP was stable against hydrolysis over 21 days and enzymatic degradation. We thus can conclude that the proposed functionalization method has high potential to tolerate harmful effects within the oral cavity and remains unchanged on the surface.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

OBJECTIVE: Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). MATERIALS AND METHODS: Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. RESULTS: Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). CONCLUSION: Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.

Topographically and Chemically Enhanced Textile Polycaprolactone Scaffolds for Tendon and Ligament Tissue Engineering.

The use of tissue engineering to address the shortcomings of current procedures for tendons and ligaments is promising, but it requires a suitable scaffold that meets various mechanical, degradation-related, scalability-related, and biological requirements. Macroporous textile scaffolds made from appropriate fiber material have the potential to fulfill the first three requirements. This study aimed to investigate the biocompatibility, sterilizability, and functionalizability of a multilayer braided scaffold. These macroporous scaffolds with dimensions similar to those of the human anterior cruciate ligament consist of fibers with appropriate tensile strength and degradation behavior melt-spun from Polycaprolactone (PCL). Two different cross-sectional geometries resulting in significantly different specific surface areas and morphologies were used at the fiber level, and a Chitosan-graft-PCL (CS-g-PCL) surface modification was applied to the melt-spun substrates for the first time. All scaffolds elicited a positive cell response, and the CS-g-PCL modification provided a platform for incorporating functionalization agents such as drug delivery systems for growth factors, which were successfully released in therapeutically effective quantities. The fiber geometry was found to be a variable that could be manipulated to control the amount released. Therefore, scaled, surface-modified textile scaffolds are a versatile technology that can successfully address the complex requirements of tissue engineering for ligaments and tendons, as well as other structures.