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20 pages, 10222 KiB  
Article
Preparation and Characterization of Novel Nanofibrous Composites Prepared by Electrospinning as Multifunctional Platforms for Guided Bone Regeneration Procedures
by Aleksandra Sierakowska-Byczek, Julia Radwan-Pragłowska, Łukasz Janus, Tomasz Galek, Natalia Radwan-Pragłowska, Karol Łysiak, Piotr Radomski and Mirosław Tupaj
Appl. Sci. 2025, 15(5), 2578; https://doi.org/10.3390/app15052578 - 27 Feb 2025
Viewed by 147
Abstract
Prosthetics, a rapidly advancing field in dentistry, aims to improve patient comfort and aesthetics by addressing the challenge of replacing missing teeth. A critical obstacle in dental implantation is the condition of the jawbone, which often necessitates reconstruction prior to implant placement. Guided [...] Read more.
Prosthetics, a rapidly advancing field in dentistry, aims to improve patient comfort and aesthetics by addressing the challenge of replacing missing teeth. A critical obstacle in dental implantation is the condition of the jawbone, which often necessitates reconstruction prior to implant placement. Guided bone regeneration (GBR) and guided tissue regeneration (GTR) techniques utilize membranes that act as scaffolds for bone and tissue growth while serving as barriers against rapidly proliferating cells and pathogens. Commonly used membranes, such as poly(tetrafluoroethylene) (PTFE) and collagen, have significant limitations—PTFE is non-bioresorbable and requires secondary removal, while collagen lacks adequate mechanical strength and exhibits unpredictable degradation rates. To overcome these challenges, nanofiber membranes produced via electrospinning using polylactic acid (PLA) were developed. The novel composites were functionalized with bioactive additives, including periclase (MgO) nanoparticles and polydopamine (PDA), to enhance osteoblast adhesion, antibacterial properties, and tissue regeneration. This study comprehensively evaluated the biological, mechanical, and physicochemical properties of the prepared nanofibrous scaffolds. Experimental results revealed controlled degradation rates and improved hydrophilicity due to surface modifications with PDA and MgO. Moreover, the nanofibers exhibited enhanced swelling behavior, which promoted nutrient exchange while maintaining structural integrity over prolonged periods. The incorporation of bioactive additives contributed to superior osteoblast proliferation, antibacterial activity, and growth factor immobilization, supporting bone tissue regeneration. These findings suggest that the developed nanofibrous composites are a promising candidate for GBR and GTR applications, offering a balanced combination of biological activity, mechanical performance, and degradation behavior tailored for clinical use. Full article
(This article belongs to the Special Issue Cutting-Edge Developments in Prosthodontics and Dental Implants)
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Figure 1

Figure 1
<p>XRD diffractogram confirming preparation of highly crystalline periclase.</p>
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<p>Vector plot of electric field intensity for electrospinning machine for (<b>a</b>)—50 mm; (<b>b</b>)—100 mm needle-to-collector distance.</p>
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<p>Vector plot of electric field intensity for electrospinning machine for (<b>a</b>)—50 mm; (<b>b</b>)—100 mm needle-to-collector distance.</p>
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<p>Magnitude of the electric field intensity for the needle end and surroundings (front view and top view) for (<b>a</b>)—50 mm; (<b>b</b>)—100 mm needle-to-collector distance.</p>
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<p>The electric field intensity as a distance function from the needle end (y-coordinate) for 50 mm needle-to-collector distance.</p>
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<p>General scheme for biomaterials preparation and application.</p>
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<p>FT-IR spectrum of raw materials and ready nanofibers.</p>
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<p>TEM microphotographs (<b>a</b>)—periclase (<b>b</b>)—poly(dopamine).</p>
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<p>Nanofibers morphology containing periclase (×20k magnification): (<b>a</b>)—sample 2; (<b>b</b>)—sample 3; (<b>c</b>)—sample 4 (<b>d</b>)—sample 4 with extra magnification (×50k); (<b>e</b>)—sample 5; (<b>f</b>)—sample 6.</p>
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<p>EDS analysis of the GBR membrane sample 6: (<b>a</b>) pink—carbon; (<b>b</b>) blue magnesium; (<b>c</b>) yellow oxygen; (<b>d</b>) all elements marked (C, Mg, O).</p>
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<p>General scheme of GBR procedure after tooth extraction: (<b>a</b>) healthy socket with tooth; (<b>b</b>) empty socket after tooth extraction; (<b>c</b>) natural bone resorption process after tooth removal; (<b>d</b>) guided bone regeneration due to separation of cavity from epithelial cells.</p>
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<p>Swelling degree of samples 1–6.</p>
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<p>Possible consequences of tooth extraction: (<b>a</b>) tooth loss due to caries: 1—dental biofilm formation, 2—caries leading to tooth loss, 3—empty socket after extraction, 4—bone resorption; (<b>b</b>) the most promising pathway including GBR procedure: 5—bone augmentation, 6—proliferating osteoblasts, 7—successful implantation and osseofixation without biofilm formation (<b>c</b>) alternative scenario without GBR procedure—socket filling with epithelial cells (<b>d</b>) issues with osseofixation and caries of adjacent teeth due to lack of biofilm formation prevention.</p>
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<p>Quantitative cytotoxicity study on MG-63 osteosarcoma cells.</p>
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<p>Microscopic images of (<b>a</b>)—sample 5 and (<b>b</b>)—sample 6.</p>
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22 pages, 5010 KiB  
Article
Porcine Bone Extracellular Matrix Hydrogel as a Promising Graft for Bone Regeneration
by Rotem Hayam, Shani Hamias, Michal Skitel Moshe, Tzila Davidov, Feng-Chun Yen, Limor Baruch and Marcelle Machluf
Gels 2025, 11(3), 173; https://doi.org/10.3390/gels11030173 - 27 Feb 2025
Viewed by 322
Abstract
Bone defects resulting from trauma, tumors, or congenital conditions pose significant challenges for natural healing and often require grafting solutions. While autografts remain the gold standard, their limitations, such as restricted availability and donor site complications, underscore the need for alternative approaches. The [...] Read more.
Bone defects resulting from trauma, tumors, or congenital conditions pose significant challenges for natural healing and often require grafting solutions. While autografts remain the gold standard, their limitations, such as restricted availability and donor site complications, underscore the need for alternative approaches. The present research investigates the potential of porcine-derived bone extracellular matrix (pbECM) hydrogel as a highly promising bioactive scaffold for bone regeneration, comparing it to the human-derived bECM (hbECM). Porcine and human cancellous bones were decellularized and characterized in terms of their composition and structure. Further, the ECMs were processed into hydrogels, and their rheological properties and cytocompatibility were studied in vitro while their biocompatibility was studied in vivo using a mouse model. The potential of the pbECM hydrogel as a bone graft was evaluated in vivo using a rat femoral defect model. Our results demonstrated the excellent preservation of essential ECM components in both the pbECM and hbECM with more than 90% collagen out of all proteins. Rheological analyses revealed the superior mechanical properties of the pbECM hydrogel compared to the hbECM, with an approximately 10-fold higher storage modulus and a significantly later deformation point. These stronger gel properties of the pbECM were attributed to the higher content of structural proteins and residual minerals. Both the pbECM and hbECM effectively supported mesenchymal stem cell adhesion, viability, and proliferation, achieving a 20-fold increase in cell number within 10 days and highlighting their strong bioactive potential. In vivo, pbECM hydrogels elicited a minimal immunogenic response. Most importantly, when implanted in a rat femoral defect model, pbECM hydrogel had significantly enhanced bone regeneration through graft integration, stem cell recruitment, and differentiation. New bone formation was observed at an average of 50% of the defect volume, outperforming the commercial demineralized bone matrix (DBM), in which the new bone filled only 35% of the defect volume. These results position pbECM hydrogel as a highly effective and biocompatible scaffold for bone tissue engineering, offering a promising alternative to traditional grafting methods and paving the way for future clinical applications in bone repair. Full article
(This article belongs to the Special Issue Gels for Biomedical Applications)
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Graphical abstract

Graphical abstract
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<p>Decellularization process of bECM from cancellous bone tissue. Porcine (<b>A</b>) and human (<b>B</b>) bone fragments. Decellularized bECM from porcine (<b>C</b>) and human (<b>D</b>) bones. H&amp;E staining of decellularized bECM from porcine (<b>E</b>) and human (<b>F</b>) bone fragments. (<b>G</b>) DNA content in porcine and human native bones and bECM, quantified using PicoGreen assay. ** <span class="html-italic">p</span> &lt; 0.0011, **** <span class="html-italic">p</span> &lt; 0.0001. Scale bars, 200 µm.</p>
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<p>Protein composition of porcine and human bECMs. (<b>A</b>) Immunostaining of porcine and human bECMs for collagen I, collagen V, and collagen IV. Scale bars, 50 μm. (<b>B</b>,<b>C</b>) Content of the different collagen types in pbECM (<b>B</b>) and hbECM (<b>C</b>). (<b>D</b>,<b>E</b>) A more detailed analysis of the less abundant collagen types (excluding collagen type I) in pbECM (<b>D</b>) and hbECM (<b>E</b>). (<b>F</b>) Collagen percentage out of the protein content in porcine and human bECMs. (<b>G</b>) Proteoglycans content in porcine and human bECMs.</p>
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<p>Characterization of bECM from human and porcine bone tissue. SEM images of (<b>A</b>) pbECM and (<b>B</b>) hbECM, scale bars 400 nm. (<b>C</b>) Fiber diameter distribution in pbECM and hbECM, analyzed from SEM images using ImageJ 1.53r. (<b>D</b>) Fourier transform infrared spectroscopy (FTIR) spectra of the pbECM compared to hbECM. (<b>E</b>) X-ray diffraction (XRD) analysis of the human and porcine bECM compared to commercial hydroxyapatite.</p>
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<p>bECM hydrogels’ characterization. (<b>A</b>) Mechanical properties of the bECM hydrogels. (<b>Upper panel</b>): Time sweep rheological characterization, changes in G′ and G″ over time. (<b>Lower panel</b>): Frequency sweep rheological characterization, changes in G′ and G″ in ascending frequencies. Cytocompatibility of bECM hydrogels: (<b>B</b>) adherence of MSCs seeded to bECM hydrogels, and (<b>C</b>) viability over 10 days of culture, relative to the cell viability on a culture plate at day 1. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. Confocal microscope images of MSCs on the (<b>D</b>) porcine and (<b>E</b>) human bECM hydrogels 10 days post-seeding. Red: Phalloidin (Actin), blue: DAPI (DNA). Scale bars 20 µm.</p>
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<p>Biocompatibility of bECM. RAW macrophage stimulation assay in vitro: (<b>A</b>) NO secretion level, (<b>B</b>,<b>C</b>) expression of IL-1β (<b>B</b>) and TNF-α (<b>C</b>) mRNA, normalized to GAPDH. **** <span class="html-italic">p</span> &lt; 0.0001. In vivo biocompatibility study: (<b>D</b>) complete blood counts of mice following subcutaneous implantation of bECM hydrogels. Number of white blood cells (WBCs) and red blood cells (RBCs), hematocrit volume, hemoglobin concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), number of neutrophils, and lymphocytes, all plotted over three weeks following implantation. Dashed lines represent basal blood values for C57 black mice. (<b>E</b>–<b>G</b>) H&amp;E staining (scale bars 500 μm) and (<b>H</b>–<b>J</b>) immunohistochemistry analyses for F4/80 (scale bars 100 μm) of hydrogels retrieved after 22 days post-implantation.</p>
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<p>Efficacy of the pbECM hydrogel graft. (<b>A</b>) The 2D images of the micro-CT 3D reconstructed femoral condyles following treatment with pbECM hydrogel, DBM, or PBS. Images for each experimental group were chosen to demonstrate the best (<b>upper panel</b>) and worst (<b>lower panel</b>) bone repair observed from micro-CT scans. Small circles mark the defect area, which is enlarged in the large circles of each image. (<b>B</b>) Percentage of the bone volume from the total defect volume of the pbECM hydrogel graft-treated group, DBM graft-treated group, and PBS untreated control group, as derived from the micro-CT images (CTAn Micro-CT Software Version 1.17.7.2). * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Histopathological assessment of bone defect regeneration. (<b>A</b>) H&amp;E and (<b>B</b>) Masson’s trichrome histological analysis of the defect site following treatment with pbECM hydrogel, DBM, or PBS. Immunohistochemistry assessment of the regenerated defect site for (<b>C</b>) RUNX2 and (<b>D</b>) osteocalcin (OC). Scale bars: 2 mm. Small circles mark the defect area, which is enlarged in the large circles of each image. Percentage of (<b>E</b>) collagen-stained area, (<b>F</b>) RUNX2-stained area, and (<b>G</b>) OC-stained area within the femur defect site, quantified from the histological sections (QuPath software V0.5.1). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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19 pages, 8571 KiB  
Article
Molecular Diffusion and Optical Properties of Implantable Collagen Materials
by Sofya V. Atsigeida, Daria K. Tuchina, Peter S. Timashev and Valery V. Tuchin
Materials 2025, 18(5), 1035; https://doi.org/10.3390/ma18051035 - 26 Feb 2025
Viewed by 131
Abstract
The effects of optical clearing of implantable collagen materials were studied using optical clearing agents (OCAs) based on aqueous glucose solutions of various concentrations. By measuring the kinetics of the collimated transmission spectra, the diffusion D and permeability P coefficients of the OCAs [...] Read more.
The effects of optical clearing of implantable collagen materials were studied using optical clearing agents (OCAs) based on aqueous glucose solutions of various concentrations. By measuring the kinetics of the collimated transmission spectra, the diffusion D and permeability P coefficients of the OCAs of collagen materials were determined as D = (0.22 ± 0.05) × 10−6 to (1.41 ± 0.05) × 10−6 cm2/c and P = (0.55 ± 0.04) × 10−4 to (1.77 ± 0.07) × 10−4 cm/c. Studies with optical coherence tomography (OCT) confirmed that each of the OCAs used had an effect on the optical properties of collagen materials, and allowed us to quantify the group refractive indices of the collagen of various samples, which turned out to be in the range from nc = 1.476 to nc = 1.579. Full article
(This article belongs to the Special Issue Advances in Optical and Photonic Materials)
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Figure 1

Figure 1
<p>Images of the “PER uncrosslinked” (<b>a</b>–<b>d</b>) and “SPILAK” (<b>e</b>–<b>h</b>) samples in dry state (first image), after exposure to saline (second image) and after additional exposure to an aqueous glucose solution (third image) of 20% (<b>a</b>,<b>e</b>), 32% (<b>b</b>,<b>f</b>), 40% (<b>c</b>,<b>g</b>) and 50% (<b>d</b>,<b>h</b>).</p>
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<p>Scheme of the experimental installation.</p>
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<p>Images of “PER uncrosslinked” samples before (<b>a</b>) and after editing the image (<b>b</b>).</p>
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<p>The THORLABS Spectral Radar OCT System (930 nm).</p>
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<p>The collimated transmission spectra and the corresponding normalized kinetic dependences at wavelengths of 650, 700, 750 and 800 nm for the “PER uncrosslinked” samples under the action of aqueous glucose solutions of different concentrations.</p>
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<p>The collimated transmission spectra and the corresponding normalized kinetic dependences at wavelengths of 650, 700, 750 and 800 nm for the “PER uncrosslinked” samples under the action of aqueous glucose solutions of different concentrations.</p>
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<p>The collimated transmission spectra and the corresponding normalized kinetic dependences at wavelengths of 650, 700, 750 and 800 nm for the “SPILAK” samples under the action of aqueous glucose solutions of different concentrations.</p>
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<p>Changes in the collimated transmission of the “PER uncrosslinked” (<b>a</b>) and “SPILAK” (<b>b</b>) samples during their impregnation by saline.</p>
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<p>Dependences of thickness and weight of the samples on the exposure time to saline (<b>a</b>,<b>b</b>), (<b>e</b>,<b>f</b>) or deionized distilled water (<b>c</b>,<b>d</b>), (<b>h</b>,<b>g</b>), (<b>i</b>,<b>j</b>): “PER uncrosslinked” (<b>a</b>–<b>d</b>); “SPILAK” (<b>e</b>,<b>f</b>), (<b>h</b>,<b>g</b>); “PER crosslinked”(<b>i</b>,<b>j</b>).</p>
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<p>Dependences of thickness and weight of the samples on the exposure time to saline (<b>a</b>,<b>b</b>), (<b>e</b>,<b>f</b>) or deionized distilled water (<b>c</b>,<b>d</b>), (<b>h</b>,<b>g</b>), (<b>i</b>,<b>j</b>): “PER uncrosslinked” (<b>a</b>–<b>d</b>); “SPILAK” (<b>e</b>,<b>f</b>), (<b>h</b>,<b>g</b>); “PER crosslinked”(<b>i</b>,<b>j</b>).</p>
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<p>Typical OCT images of “PER uncrosslinked” samples: dry (<b>a</b>), after exposure to saline (<b>b</b>), after subsequent exposure to 20% aqueous glucose solution (<b>c</b>); dry (<b>d</b>), after exposure to saline (<b>e</b>), after subsequent exposure to 32% aqueous glucose solution (<b>f</b>).</p>
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<p>The OCT signal on the depth of “PER uncrosslinked” samples exposed to OCA: 20% (<b>a</b>) and 32% (<b>b</b>) glucose solution.</p>
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<p>Typical OCT images of “SPILAK” samples: dry (<b>a</b>), after exposure to saline (<b>b</b>), after subsequent exposure to 40% glucose solution (<b>c</b>); dry (<b>d</b>); after exposure to saline (<b>e</b>); after subsequent exposure to 50% glucose solution (<b>f</b>).</p>
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<p>The OCT signal on the depth of the “SPILAK” samples exposed to OCA: 40% (<b>a</b>) and 50% (<b>b</b>) glucose solution.</p>
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<p>The OCT signals averaged within the window (<a href="#materials-18-01035-f009" class="html-fig">Figure 9</a>a) depending on the scanning depth for the “PER uncrosslinked” samples before and after the action of OCA (20% glucose) for 30 min. Straight lines present the slopes of these dependencies.</p>
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17 pages, 9694 KiB  
Article
Novel Soybean Oil-Based 3D Printed Resin Membrane Used for Guided Bone Regeneration in Calvaria Bone Critical-Size Defects: A Microtomographic and Histologic Study in Rats
by Eduardo Pires Godoy, Letícia Gabriela Artioli, Daniele Botticelli, Fabrizio Nicoletti, Leonardo Dassatti, Mario Bragaglia, Francesca Nanni, Samuel Porfirio Xavier and Erick Ricardo Silva
Appl. Sci. 2025, 15(4), 2184; https://doi.org/10.3390/app15042184 - 18 Feb 2025
Viewed by 298
Abstract
Background: Osseointegrated implants are essential for rehabilitating edentulous patients, but critical bone defects remain challenging. Guided bone regeneration (GBR) with barrier membranes is an effective approach. This study evaluated a 3D printed membrane made from acrylated epoxidized soybean oil (AESO) combined with a [...] Read more.
Background: Osseointegrated implants are essential for rehabilitating edentulous patients, but critical bone defects remain challenging. Guided bone regeneration (GBR) with barrier membranes is an effective approach. This study evaluated a 3D printed membrane made from acrylated epoxidized soybean oil (AESO) combined with a xenogeneic graft for GBR in critical-size defects. Methods: Forty-eight male Sprague Dawley rats (150 g) were assigned to four groups: a negative control group (NC, blood clot only), a positive control group (PC, biomaterial without membrane), a negative test group (NT, blood clot with membrane), and a positive test group (PT, biomaterial with membrane). Results: The PT group showed the highest bone volume and superior bone maturation compared to the other groups. Bone quality parameters (Tb.N, Tb.Th) indicated enhanced maturation in the groups using the membrane. A histological analysis confirmed centripetal bone formation. Conclusion: The AESO-based membrane provided mechanical support and controlled resorption, addressing collagen membrane limitations. Its combination with the GTO® graft material enhanced osteoconduction, bone formation, and bone quality, highlighting its potential for complex bone defect reconstructions. Full article
(This article belongs to the Section Applied Dentistry and Oral Sciences)
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Figure 1
<p>Schematic representation of the study design. Each group, composed of randomly selected animals, received one type of treatment.</p>
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<p>Schematic drawing of the membrane printed from AESO with dimensions of 1 mm thickness, 10 mm chord, and 3 mm height.</p>
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<p>Surgical photographs of the experimental groups. (<b>a</b>) Negative control group. (<b>b</b>) Positive control group. (<b>c</b>) Negative test group. (<b>d</b>) Positive test group.</p>
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<p>Micro-CT results. The results are expressed as mean and standard deviation. (<b>a</b>) Bone volume (BV-mm<sup>3</sup>). (<b>b</b>) New bone formation (%BV/TV, threshold, 70–100). (<b>c</b>) Residual graft (%BV/TV, threshold, 100–255). Vertical bars indicate standard deviation. Horizontal bars indicate statistical differences with values of * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Three-dimensional reconstructed micro-CT images of negative control group (NC), positive control group (PC), negative test group (NT), and positive test group (PT) samples at 4 weeks and 8 weeks post-surgery. The gray color represents newly formed bone, and the blue color represents the remaining graft granules. These images were obtained using CT Analyzer software.</p>
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<p>Quality of the newly formed bone. The results are expressed as mean and standard deviation. (<b>a</b>) Trabecular number (Tb.N). (<b>b</b>) Trabecular thickness (Tb.Th). (<b>c</b>) Trabecular separation (Tb.Sp). Vertical bars indicate standard deviation. Horizontal bars indicate statistical differences with values of * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>High-magnification 3D reconstructions of bone defect repair in the negative control group (NC) and negative test group (NT) at 4 and 8 weeks, showing newly formed bone. These images were obtained using CT Analyzer software.</p>
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<p>High-magnification 3D reconstructions of bone defect repair in the positive control group (PC) and positive test group (PT) at 4 and 8 weeks, showing newly formed bone. These images were obtained using CT Analyzer software.</p>
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<p>Photomicrographs at 4 and 8 weeks of repair at 16x magnification. NC (negative control group). PC (positive control group). NT (negative test group). PT (positive test group). Alizarin red and Stevenel’s blue.</p>
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<p>Photomicrographs at 100× magnification illustrating bone formation originating from the walls of the critical-size bone defect, indicated by yellow arrows, in the negative control (NC) and negative test (NT) groups at 4 and 8 weeks. Stained with Stevenel’s blue and alizarin red.</p>
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<p>Photomicrographs at 100× magnification illustrating bone formation originating from the walls of the critical-size bone defect, indicated by yellow arrows, and bone formation over the graft granules, represented by green arrows, in the positive control (PC) and positive test (PT) groups at 4 and 8 weeks. Stevenel’s blue and alizarin red staining.</p>
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27 pages, 6858 KiB  
Article
Biofunctionalization of Collagen Barrier Membranes with Bone-Conditioned Medium, as a Natural Source of Growth Factors, Enhances Osteoblastic Cell Behavior
by Harshitha Ashoka Sreeja, Emilio Couso-Queiruga, Clemens Raabe, Vivianne Chappuis and Maria B. Asparuhova
Int. J. Mol. Sci. 2025, 26(4), 1610; https://doi.org/10.3390/ijms26041610 - 13 Feb 2025
Viewed by 377
Abstract
A key principle of guided bone regeneration (GBR) is the use of a barrier membrane to prevent cells from non-osteogenic tissues from interfering with bone regeneration in patients with hard tissue deficiencies. The aim of the study was to investigate whether the osteoinductive [...] Read more.
A key principle of guided bone regeneration (GBR) is the use of a barrier membrane to prevent cells from non-osteogenic tissues from interfering with bone regeneration in patients with hard tissue deficiencies. The aim of the study was to investigate whether the osteoinductive properties of bone-conditioned medium (BCM) obtained from cortical bone chips harvested at the surgical site can be transferred to a native bilayer collagen membrane (nbCM). BCM extracted within 20 or 40 min, which corresponds to a typical implant surgical procedure, and BCM extracted within 24 h, which corresponds to BCM released from the autologous bone chips in situ, contained significant and comparable amounts of TGF-β1, IGF-1, FGF-2, VEGF-A, and IL-11. Significant (p < 0.001) quantities of BMP-2 were only detected in the 24-h BCM preparation. The bioactive substances contained in the BCM were adsorbed to the nbCMs with almost 100% efficiency. A fast but sequential release of all investigated proteins occurred within 6–72 h, reflecting their stepwise involvement in the natural regeneration process. BCM-coated nbCM significantly (p < 0.05) increased the migratory, adhesive, and proliferative capacity of primary human bone-derived cells (hBC), primary human periodontal ligament cells (hPDLC), and an osteosarcoma-derived osteoblastic cell line (MG-63) compared to cells cultured on BCM-free nbCM. The high proliferative rates of MG-63 cells cultured on BCM-free nbCM were not further potentiated by BCM, indicating that BCM-coated nbCM has no detrimental effects on cancer cell growth. BCM-coated nbCM caused significant (p < 0.05) induction of early osteogenic marker gene expression and alkaline phosphatase activity, suggesting an important role of BCM-functionalized nbCM in the initiation of osteogenesis. The 24-h BCM loaded on the nbCM was the only BCM preparation that caused significant induction of late osteogenic marker gene expression. Altogether, our data define the pre-activation of collagen membranes with short-term-extracted BCM as a potential superior modality for treating hard tissue deficiencies via GBR. Full article
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Figure 1
<p>Release of various growth factors and cytokines involved in bone metabolism from cortical bone. Enzyme-linked immunosorbent assay (ELISA) quantification of TGF-β1, IGF-1, FGF-2, VEGF-A, IL-11, and BMP-2 proteins contained in bone-conditioned medium (BCM) extracted from cortical bone chips with Ringer’s solution (RS). BCM was collected at 20 min, 40 min, and 24 h. RS not containing bone particles represents the control (Ctrl) and exhibits no detectable levels of the proteins tested. Means ± SD from four independent BCM preparations from each type and significant differences to the control, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, are shown.</p>
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<p>Adsorption and release of TGF-β1 (<b>a</b>), IGF-1 (<b>b</b>), FGF-2 (<b>c</b>), VEGF-A (<b>d</b>), IL-11 (<b>e</b>), and BMP-2 (<b>f</b>) from nbCM coated with either the respective recombinant (r) protein or BCM preparation. The nbCM was incubated for 10 min at room temperature in either RS containing the respective recombinant protein at the average concentration measured in the BCM preparations (cf. <a href="#ijms-26-01610-f001" class="html-fig">Figure 1</a>) or each of the three BCM (20 min, 40 min, and 24 h). Hydration of the nbCM with RS was used as a control (Ctrl). Protein quantifications were performed by using colorimetric ELISA assays. Tables (a–f) represent: (1) quantifications of adsorbed protein (in percent); (2) total protein release (expressed as percent of adsorbed protein) from nbCM for a 4-day period; (3) the time point at which the highest protein release was observed (peak of release); (4) ELISA quantifications of the protein released before (and including) the peak expressed as percent of the total protein release for the entire test period (taken as 100%); (5) ELISA quantifications of the protein released after the peak until day 4 and expressed as in (4). Means ± SD from three independent experiments and significant differences (<span class="html-italic">p</span> &lt; 0.05) between the experimental groups are shown. Significance was indicated with the following symbols: # denotes significantly higher than recombinant protein; <span>$</span> denotes significantly higher than 20 min BCM; † denotes significantly higher than 40 min BCM; ‡ denotes significantly higher than 24 h BCM. Graphs represent the results from ELISA quantifications of the proteins measured in conditioned RS collected from the nbCM at the indicated time points over a 4-day period. Data represent means ± SD from three independent experiments. Significant differences between experimental groups, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Induced migration capacity of osteoblast-like cell lines toward BCM-coated nbCM. Migration of primary human bone-derived cells (hBC) (<b>a</b>,<b>b</b>), primary human periodontal ligament cells (hPDLC) (<b>c</b>,<b>d</b>), and an osteosarcoma-derived immortal cell line (MG-63) (<b>e</b>,<b>f</b>) toward nbCMs coated with different BCM preparations (20 min, 40 min, or 24 h) was evaluated by a transwell migration assay utilizing ThinCert<sup>®</sup> transwell PET membrane supports with 8 μm pore size. nbCM hydrated with RS was used as a control (Ctrl). (<b>a</b>,<b>c</b>,<b>e</b>) Representative images of fixed and stained cells that have migrated to the lower side of the filter in each of the experimental groups. Scale bar, 500 μm. (<b>b</b>,<b>d</b>,<b>f</b>) Quantification of the cell migration using the Image J software (version 1.50) measuring the area on the lower side of the filter covered with migrated cells. Data represent means ± SD from four independent experiments performed with (1) two independent BCM preparations, each used with two different cell donors for each of the two primary cell types, hBC and hPDLC, and (2) four independent BCM preparations used with the MG-63 cell line. Significant differences to the control, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Increased expression of adhesive marker genes in hBCs (<b>a</b>), hPDLCs (<b>b</b>), and MG-63 cells (<b>c</b>) grown on BCM-free nbCM (Ctrl) or nbCMs coated with either 20 min BCM, 40 min BCM, or 24 h BCM. Cells were cultured on the respective nbCM for 6 h followed by an extensive wash for complete removal of nonadherent cells from the membranes before total RNA was isolated and analyzed for the expression of adhesive marker genes (FN1, VCL, CD44, and ICAM1) by qRT-PCR. Values normalized to GAPDH are expressed relative to the values of control cells. Data represent means ± SD from four independent experiments performed with (1) two independent BCM preparations, each used with two different cell donors for each of the primary cell types, hBC and hPDLC, and (2) four independent BCM preparations used with the MG-63 cell line. Significant differences to the respective controls, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05. Morphological appearance and filamentous actin (F-actin) formation in hBCs, hPDLCs, and MG-63 cells, which, after initial attachment on control or BCM-coated nbCMs for 6 h, were detached and re-seeded on regular cell culture-treated plastic dishes for 24 h (<b>d</b>). Re-attached cells from all tested conditions were subjected to F-actin immunostaining using Alexa Fluor 488-labeled phalloidin (green). The cell nuclei were localized via DAPI co-stain (blue); a bright field (BF) image is also shown. Scale bar, 500 µm.</p>
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<p>(<b>a</b>–<b>c</b>) Proliferation rates of hBCs (<b>a</b>), hPDLCs (<b>b</b>), and MG-63 cells (<b>c</b>) grown on BCM-free nbCM (Ctrl) or nbCMs coated with either 20 min, 40 min, or 24 h BCM preparation were assessed by trypan blue dye-exclusion cell counting performed in a Countess™ II instrument on days 1, 3, 6, and 9 post-seeding. Data represent means ± SD from four independent experiments performed with (1) two independent BCM preparations, each used with two different cell donors for each of the primary cell types, hBC and hPDLC, and (2) four independent BCM preparations used with the MG-63 cell line. Significant differences to control cells at each individual time point unless otherwise indicated, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05. (<b>d</b>–<b>f</b>) Increased expression of proliferative marker genes in hBCs (<b>d</b>), hPDLCs (<b>e</b>), and MG-63 cells (<b>f</b>) grown on control or BCM-coated nbCMs. Cells were grown in the four tested conditions for 1 and 3 days before total RNA was isolated and analyzed for the expression of proliferative marker genes (MYBL2, BUB1, PLK1, and MKI67) by qRT-PCR. Values normalized to GAPDH are expressed relative to the values of control cells at day 1 (1d). Data represent means ± SD from four independent experiments performed with (1) two independent BCM preparations, each used with two different cell donors for each of the primary cell types, hBC and hPDLC, and (2) four independent BCM preparations used with the MG-63 cell line. Significant differences to the respective controls at day 1 unless otherwise indicated, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>(<b>a</b>–<b>c</b>) Increased expression of early osteogenic marker genes in hBCs (<b>a</b>), hPDLCs (<b>b</b>), and MG-63 cells (<b>c</b>) grown on control or BCM-coated nbCMs. Cells were grown in the four tested conditions for 3 days before total RNA was isolated, purified, and analyzed for the expression of COL1A1, SPP1, RUNX2, and ALPL osteogenic markers by qRT-PCR. Values normalized to GAPDH are expressed relative to the values of control cells. Data represent means ± SD from four independent experiments performed with (1) two independent BCM preparations, each used with two different cell donors for each of the primary cell types, hBC and hPDLC, and (2) four independent BCM preparations used with the MG-63 cell line. Significant differences to the respective controls unless otherwise indicated, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05. (<b>d</b>) Increased alkaline phosphatase (ALP) activity in hBCs, hPDLCs, and MG-63 cells grown on control or BCM-coated nbCMs. Cells were grown as in (<b>a</b>–<b>c</b>) before ALP activity in the cell culture supernatants was measured by a fluorometric analysis based on the hydrolysis of 4-methylumbelliferyl phosphate by the ALP into the fluorescent product 4-methylumbelliferone. Data and statistical significance are expressed as in (<b>a</b>–<b>c</b>).</p>
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<p>Differential expression of intermediate and late osteogenic marker genes in osteoblast-like cell types grown on nbCM coated with short- versus long-term extracted BCM. hBCs (<b>a</b>), hPDLCs (<b>b</b>), and MG-63 cells (<b>c</b>) were grown on control or BCM-coated nbCMs for 3 days before total RNA was extracted, purified, and analyzed for the expression of DLX5, IBSP, BGLAP2, and PHEX osteogenic markers by qRT-PCR. Values normalized to GAPDH are expressed relative to the values of control cells. Data represent means ±SD from four independent experiments performed with (1) two independent BCM preparations, each used with two different cell donors for each of the primary cell types, hBC and hPDLC, and (2) four independent BCM preparations used with the MG-63 cell line. Significant differences to the respective controls unless otherwise indicated, *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05.</p>
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13 pages, 1644 KiB  
Article
Dental Implant Rehabilitation of Posterior Maxillary Edentulism via Sinus Augmentation Using the Lateral Window Technique: A Retrospective Analysis of 289 Implants Followed Up for 15 Years
by Alper Sağlanmak, Volkan Arısan, Cüneyt Karabuda and Hakan Özyuvacı
J. Funct. Biomater. 2025, 16(2), 65; https://doi.org/10.3390/jfb16020065 - 13 Feb 2025
Viewed by 485
Abstract
The aim of this study was to analyze the marginal bone loss and survival of implants in the augmented sinus area via the lateral window approach. The effect of sinus membrane perforation as well as splinting of the upper structure was analyzed. Two [...] Read more.
The aim of this study was to analyze the marginal bone loss and survival of implants in the augmented sinus area via the lateral window approach. The effect of sinus membrane perforation as well as splinting of the upper structure was analyzed. Two hundred and eighty-nine implants were placed in the sinus areas augmented with xenografts and collagen membranes in 101 patients. Clinical and radiographic data were obtained during recall visits. The Marginal Bone Loss (MBL) and Cumulative Survival Rate (CSR) were evaluated. The mean follow-up period was 12.4 years (range: 12 to 182 months). During the follow-up period, 19 implants were lost, yielding a 92.93% survival rate. No significant models for any of the covariates were found in terms of implant survival (p = 0.08). Similarly, no significant differences were observed between intact and perforated sinuses (p = 0.41) or between splinted or single standing implants (p = 0.11). The overall MBL reached 1.80 ± 0.56 mm at 15 years, and no significant differences were detected between any particular years (p = 0.12). Dental implant rehabilitation of the posterior maxilla via sinus augmentation using the lateral window technique is safe, effective and provides a high long-term implant survival with minimal prosthetic complications. Full article
(This article belongs to the Special Issue Recent Advances in Bone Graft Materials)
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<p>(<b>a</b>–<b>f</b>) Surgical steps of sinus augmentation via lateral window approach. (<b>b</b>) Demarcation of the lateral wall using the trap door technique. (<b>c</b>) Dissection and elevation of the Schneiderian membrane using the specially designed instruments. (<b>d</b>) Application of the graft material beneath the elevated sinus membrane (<b>e</b>) Positioning of the collagen membrane over the lateral window. (<b>f</b>) Primary flap closure with 3.0 silk sutures.</p>
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<p>MBL around implants placed into perforated and intact sinus membrane areas. Vertical bars represent the standard deviation.</p>
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<p>Survival curves of implant failures in the perforated and intact sinuses. Kaplan–Meier survival analysis. Log-rank test, <span class="html-italic">p</span> = 0.41. Symbols represent failed implants and implants lost during follow-up. Vertical bars represent the cumulative survival probability.</p>
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<p>Survival curves of implant failures in the splinted and single standing implants. Kaplan–Meier survival analysis. Log-rank test <span class="html-italic">p</span> = 0.11. Symbols represent failed implants and implants lost during follow-up. Vertical bars represent the cumulative survival probability.</p>
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24 pages, 52987 KiB  
Article
Differential Remodelling of Endometrial Extracellular Matrix in the Non-Pregnant Uterus of Lagostomus maximus as a Potential Mechanism Underlying Embryonic Death
by Francisco Acuña, Gisela Soledad Gualdoni, Francisco Rivollier, Camila Barril, Enrique Leo Portiansky, Claudio Gustavo Barbeito and Elisa Cebral
Animals 2025, 15(4), 542; https://doi.org/10.3390/ani15040542 - 13 Feb 2025
Viewed by 391
Abstract
During development, the remodelling of fibrillar components of the uterine extracellular matrix (ECM), mediated by the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), plays an essential role in embryonic survival. Previously, we observed that in the plains viscacha (Lagostomus maximus), [...] Read more.
During development, the remodelling of fibrillar components of the uterine extracellular matrix (ECM), mediated by the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), plays an essential role in embryonic survival. Previously, we observed that in the plains viscacha (Lagostomus maximus), only caudal implantation sites (IS) contain viable embryos, whereas embryos at cranial and middle IS die and are reabsorbed. The objective of this study was to analyse the distribution and expression of key components of the endometrial ECM, including fibrillar collagens, MMPs 2 and 9, and TIMPs 1 and 2, in three uterine segments (US) of the non-pregnant adult viscachas. In sections from three US, we observed a significant craniocaudal increase in collagen fibres (Van Gieson and Picrosirius red staining) and elastic fibres (Verhoeff-Van Gieson trichrome staining), along with the immunolabelling levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 (immunohistochemistry). Zymography revealed similar gelatinolytic activity of MMP-2 in the three US but higher than the MMP-9 activity. However, MMP-9 activity in the caudal segment was significantly higher than that in the cranial and middle ones. These findings suggest that uterine ECM variations along the craniocaudal axis may contribute to uterine remodelling processes that regulate embryonic survival during gestation. Full article
(This article belongs to the Section Animal Reproduction)
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Figure 1
<p>Schematic representation of the non-pregnant uterine horns of <span class="html-italic">L. maximus</span>. (<b>A</b>) On the right, macroscopic view of the cranial, middle, and caudal uterine segments. Scale bar: 1 cm. (<b>B</b>) Cross-sectional view of one of the uterine segments. The endometrium is circumscribed by a dashed line, with the superficial and deep endometrial zones identified.</p>
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<p>Histology of the endometrium of the cranial (<b>A</b>), middle (<b>B</b>), and caudal (<b>C</b>) uterine segments of non-pregnant <span class="html-italic">L. maximus</span>. Haematoxylin-eosin. Scale bar: 500 µm. Abbreviations: En, endometrium; Le, luminal epithelium; Lp, lamina propria; Ut, uterine glands.</p>
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<p>Representative images of sections from the three uterine segments of non-pregnant <span class="html-italic">L. maximus</span>, stained with Van Gieson to identify collagen fibres in the connective tissue of the endometrium (solid line). (<b>A</b>) Image showing collagen fibres homogeneously distributed throughout the thickness of the endometrium in the cranial uterine segment. (<b>B</b>,<b>C</b>) Images showing collagen fibres predominantly distributed in the deep region (asterisk) of the endometrium in the middle (<b>B</b>) and caudal (<b>C</b>) segments. (<b>D</b>) Percentage (mean and standard deviation (SD) of collagen fibre area stained with Picrosirius in the superficial and deep endometrial zones of the three uterine segments. Significance: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. Scale bar: 200 µm.</p>
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<p>Representative images of histological sections from the three uterine segments of non-pregnant <span class="html-italic">L. maximus</span>, stained with Picrosirius red and observed under polarised light microscopy to identify collagen fibres of types I and III in the connective tissue of the endometrium. (<b>A</b>–<b>C</b>) Images of collagen fibres in the deep connective tissue of the endometrium from the three uterine segments. Type I collagen (red-yellow, blue arrowhead) and type III collagen (green, white arrowhead). (<b>D</b>) Percentage (mean and SD) of staining area for type I and III collagen in the deep connective tissue of the endometrium across the three uterine segments. (<b>E</b>) Type I/III collagen ratio in the connective tissue of the deep endometrial zone in the three uterine segments. Significance: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 200 µm.</p>
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<p>Representative images of sections from the three uterine segments of non-pregnant <span class="html-italic">L. maximus</span>, stained with Verhoff-Van Gieson to visualise elastic fibres in the connective tissue of the endometrium. (<b>A</b>–<b>C</b>) Localisation (asterisk) of elastic fibres in the superficial endometrium in the cranial (<b>A</b>), middle (<b>B</b>), and caudal (<b>C</b>) uterine segments. (<b>D</b>) Percentage (mean and SD) of staining area for elastic fibres in the superficial endometrium across the three analysed segments. Significance: ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 500 µm. Abbreviations: Le, luminal epithelium.</p>
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<p>Immunoexpression and semi-quantification of MMP-2 and MMP-9 in the superficial and deep endometrial zones of the cranial, middle, and caudal uterine segments of <span class="html-italic">L. maximus</span>. (<b>A</b>–<b>C</b>) Immunostaining of MMP-2 in the luminal epithelial tissue and connective tissue of the superficial endometrium from the cranial (<b>A</b>), middle (<b>B</b>), and caudal (<b>C</b>) uterine segments. Inserts show high magnification of the epithelium. (<b>D</b>) Quantification of the percentage of immunostained area of MMP-2 in the luminal epithelium and connective tissue of the superficial endometrium across the three uterine segments. (<b>E</b>–<b>G</b>) Immunostaining MMP-2 in the glandular epithelial tissue and connective tissue of the deep endometrium in the cranial (<b>E</b>), middle (<b>F</b>), and caudal (<b>G</b>) uterine segments. Inserts show high magnification of the epithelium. (<b>H</b>) Percentage of immunostained area of MMP-2 in the glandular epithelium and connective tissue of the deep endometrium across the three uterine segments. (<b>I</b>–<b>K</b>) Immunostaining of MMP-9 in the luminal epithelial tissue and connective tissue of the superficial endometrium in the cranial (<b>I</b>), middle (<b>J</b>), and caudal (<b>K</b>) uterine segments. (<b>L</b>) Percentage of immunostained area of MMP-9 in the aforementioned tissues in the superficial endometrium of the three analysed uterine segments. (<b>M</b>–<b>O</b>) Immunostaining of MMP-9 in the glandular epithelial tissue and connective tissue of the deep endometrium in the cranial (<b>M</b>), middle (<b>N</b>), and caudal (<b>O</b>) uterine segments. (<b>P</b>) Percentage of immunostained area of MMP-9 in the aforementioned tissues in the deep endometrium of the three analysed segments. Significance: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 200 µm (<b>A</b>–<b>C</b>,<b>E</b>–<b>G</b>,<b>I</b>–<b>K</b>,<b>M</b>–<b>O</b>), 20 µm (Inserts). Abbreviations: Ct, connective tissue; Ge, glandular epithelium; Le, luminal epithelium.</p>
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<p>Immunostaining and semi-quantification of TIMP-1 and TIMP-2 in the superficial and deep endometrial zones of the cranial, middle, and caudal uterine segments of <span class="html-italic">L. maximus</span>. (<b>A</b>–<b>C</b>) Immunostaining of TIMP-1 in the luminal epithelial tissue, connective tissue, and blood vessels in the superficial endometrium of the cranial (<b>A</b>), middle (<b>B</b>), and caudal (<b>C</b>) uterine segments. Inserts show high magnification of the epithelium. (<b>D</b>) Percentage of immunostained area of TIMP-1 in the luminal epithelial and connective tissues of the superficial endometrium across the three analysed segments. (<b>E</b>–<b>G</b>) Immunostaining of TIMP-1 in the glandular epithelial tissue, connective tissue, and blood vessels in the deep endometrium of the cranial (<b>E</b>), middle (<b>F</b>), and caudal (<b>G</b>) uterine segments. (<b>H</b>) Percentage of immunostained area of TIMP-1 in the aforementioned tissues in the deep endometrium across the three analysed segments. (<b>I</b>–<b>K</b>) Immunostaining of TIMP-2 in the luminal epithelial tissue, connective tissue, and blood vessels in the superficial endometrium of the cranial (<b>I</b>), middle (<b>J</b>), and caudal (<b>K</b>) uterine segments. Inserts show high magnification of the epithelium (<b>L)</b> Percentage of immunostained area of TIMP-2 in the aforementioned tissues in the superficial endometrium across the three analysed segments. (<b>M</b>–<b>O</b>) Immunostaining of TIMP-2 in the glandular epithelial tissue, connective tissue, and blood vessels in the deep endometrium of the cranial (<b>M</b>), middle (<b>N</b>), and caudal (<b>O</b>) uterine segments. (<b>P</b>) Percentage of immunostained area of TIMP-2 in the aforementioned tissues in the deep endometrium across the three analysed segments. Significance: ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. Scale bar: 200 µm (<b>A</b>–<b>C</b>,<b>E</b>–<b>G</b>,<b>I</b>–<b>K</b>,<b>M</b>–<b>O</b>), 20 µm (Inserts). Abbreviations: Bv, blood vessels; Ct, connective tissue; Ge, glandular epithelium; Le, luminal epithelium.</p>
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<p>Total content of MMPs and their inhibitors and balances between MMPs and TIMPs in the endometrium of the cranial, middle, and caudal uterine segments of <span class="html-italic">L. maximus</span>. (<b>A</b>,<b>C</b>) Total content (mean percentage of immunostained area ± SD) of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the superficial and deep endometrium of the cranial, middle, and caudal uterine segments. (<b>B</b>) Balance between MMPs expressed as the mean MMP-2/MMP-9 index (±SD). (<b>D</b>) Mean TIMP-1/TIMP-2 index (±SD). (<b>E</b>) Mean MMPs/TIMPs indices (±SD). Significance: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ## <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Gelatinolytic activity of MMP-2 and MMP-9 in cranial, middle, and caudal uterine segments of non-pregnant <span class="html-italic">L. maximus</span>. (<b>A</b>) Representative zymography of MMP-2 and MMP-9 in endometrial tissue samples from cranial (Cr), middle (M), and caudal (Ca) uterine segments, showing the active forms of MMP-9 in the 95 kDa molecular weight range and MMP-2 in the 50–60 kDa range. (<b>B</b>) Figure shows endometrial MMP-2 and MMP-9 activity levels, expressed as average arbitrary units (AUs) and standard deviation (SD) of active gelatinolytic MMP bands, as well as the ratio of MMP-2/MMP-9 activity in the cranial, middle, and caudal segments of the endometrium. Significance: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, ### <span class="html-italic">p</span> &lt; 0.001.</p>
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20 pages, 10627 KiB  
Article
Bone Fillers with Balance Between Biocompatibility and Antimicrobial Properties
by Bogdan Valeriu Sorca, Durmuş Alpaslan Kaya, Madalina Georgiana Albu Kaya, Marius Enachescu, Daniela-Madalina Ghetu, Laura-Bianca Enache, Iulian Boerasu, Alina Elena Coman, Laura Cristina Rusu, Rodica Constantinescu and Irina Titorencu
Biomimetics 2025, 10(2), 100; https://doi.org/10.3390/biomimetics10020100 - 10 Feb 2025
Viewed by 632
Abstract
Millions of people request bone regeneration every year, and the market for bone grafting materials has a positive trend. The most used biomaterials applied to replace and regenerate bone are based on collagen and different types of ceramics in order to mimic natural [...] Read more.
Millions of people request bone regeneration every year, and the market for bone grafting materials has a positive trend. The most used biomaterials applied to replace and regenerate bone are based on collagen and different types of ceramics in order to mimic natural bone matrix. However, there are a lot of implant-associated infections after surgery, or the implants are rejected because of reduced biocompatibility, and this is why the research into graft bone materials is still a challenge. This study aims to develop and characterize novel biomimetic bone fillers which have simultaneously both antimicrobial properties and biocompatibility with human bone marrow—derived mesenchymal stem cells (BMSCs). Type I collagen and calcium triphosphate in a ratio of 1:1 were used as a control, according to our previous studies, and ZnO, functionalized with different percentages of Satureja thymbra L. essential oils, was added as an antimicrobial, promoting bone growth, mineralization, and formation. The bone fillers were obtained by freeze-drying in spongious forms and characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), water uptake, biodegradability over time, antimicrobial activity against Staphylococcus aureus and Escherichia coli and viability and proliferation of human BMSCs. The graft material showed a higher porosity with interconnected pores, gradual resorption over time and a balance between antimicrobial properties and biocompatibility and was chosen as an ideal bone filler. Full article
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<p>Water uptake of composite sponges L1–L6.</p>
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<p>Biodegradation of composite sponges L1–L6.</p>
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<p>FT-IR spectra of (<b>a</b>) a sponge composite (L3) and its main components and (<b>b</b>) overlap of FT-IR spectra for L1, L2 and L3 composites.</p>
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<p>SEM of sponge composites: (<b>a</b>) collagen control; (<b>b</b>) L1; (<b>c</b>) L2; (<b>d</b>) L6; (<b>e</b>) EDX map of L6 sponge composite (magnification ×300).</p>
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<p>SEM of sponge composites: (<b>a</b>) collagen control; (<b>b</b>) L1; (<b>c</b>) L2; (<b>d</b>) L6; (<b>e</b>) EDX map of L6 sponge composite (magnification ×300).</p>
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<p>Compressive stress–strain curve of L1–L6 sponge composites.</p>
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<p>Antibacterial activity of sponge composites L1–L6 against <span class="html-italic">E.coli</span> and <span class="html-italic">S. aureus</span> measured by disc diffusion method: (<b>a</b>) images of samples and (<b>b</b>) size of inhibition zone, mm.</p>
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<p>Antibacterial activity of sponge composites L1–L6 against <span class="html-italic">E.coli</span> and <span class="html-italic">S. aureus</span> measured by disc diffusion method: (<b>a</b>) images of samples and (<b>b</b>) size of inhibition zone, mm.</p>
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<p>Evaluation of cytotoxic effect of the sponge composites using the XTT assay. The viability of human BMSCs cultured for 5 days on the samples was assessed. Data represent mean ± SD, expressed as percentage of the control viability (L1 sample).</p>
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<p>Determination of the capacity of the sponge composites to sustain human BMSC proliferation. Data represent mean ± SD of the DNA content, expressed as arbitrary units.</p>
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<p>Representative images of eosin-Hoechst staining of the sponge composites seeded with human BMSCs (5 days post-seeding)—peripheral region of the composites (40× magnification). Nuclei stained with Hoechst (blue) and cells cytoplasm stained with eosin (red).</p>
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<p>Representative images of eosin-Hoechst staining of the sponge composites seeded with human BMSCs (5 days post-seeding)—central region of the composites (40× magnification). Nuclei stained with Hoechst (blue) and cells cytoplasm stained with eosin (red).</p>
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<p>Representative images of human BMSCs stained for F-actin on sponge scaffolds after 5 days of culture—peripheral region of the composites. Fluorescent images of F-actin (green), nuclei (blue), merged images and phase contrast images are shown. Magnification: 40×.</p>
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<p>Representative images of human BMSCs stained for F-actin on sponge scaffolds after 5 days of culture—central region of the composites. Fluorescent images of F-actin (green), nuclei (blue), merged images and phase contrast images are shown. Magnification: 40×.</p>
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<p>ALP activity of human BMSCs seeded on the sponge composites. ALP activity was measured after 5 days of culture and normalized by DNA content of the cells. Values reported as mean ± SD represent nmols p-nitrophenol/ng DNA/min.</p>
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19 pages, 1011 KiB  
Systematic Review
Riboflavin-Mediated Photodynamic Therapy in Periodontology: A Systematic Review of Applications and Outcomes
by Jakub Fiegler-Rudol, Maciej Łopaciński, Artur Los, Dariusz Skaba and Rafał Wiench
Pharmaceutics 2025, 17(2), 217; https://doi.org/10.3390/pharmaceutics17020217 - 7 Feb 2025
Viewed by 429
Abstract
Background: Riboflavin (vitamin B2) has emerged as a promising photosensitizer in photodynamic therapy (PDT) due to its strong absorption of blue light and favourable safety profile. This systematic review aims to evaluate the efficacy of riboflavin-mediated PDT in periodontology, specifically [...] Read more.
Background: Riboflavin (vitamin B2) has emerged as a promising photosensitizer in photodynamic therapy (PDT) due to its strong absorption of blue light and favourable safety profile. This systematic review aims to evaluate the efficacy of riboflavin-mediated PDT in periodontology, specifically examining its antimicrobial effects and potential to improve clinical outcomes compared to conventional or other PDT-based treatments. Methods: A systematic review was conducted following PRISMA guidelines. A comprehensive literature search was performed in PubMed/Medline, Embase, Scopus, and the Cochrane Library. Studies published in English within the last 10 years were considered, where riboflavin served as the primary photosensitizer for dental treatments. Data extraction focused on study design, photosensitizer concentration, light source parameters, and clinical or microbiological outcomes. The risk of bias was assessed independently by two reviewers using a predefined scoring system. Results: Ten studies met the inclusion criteria, all demonstrating a low risk of bias. Riboflavin-mediated PDT consistently reduced microbial biofilms and pathogen viability in periodontitis, peri-implantitis, and endodontic models. Although some studies reported slightly lower efficacy compared to chlorhexidine or toluidine blue–based PDT, riboflavin-mediated PDT exhibited advantages such as minimal staining, low cytotoxicity, and enhanced collagen crosslinking. However, most studies were in vitro or small-scale clinical trials, limiting conclusions on long-term effectiveness. Conclusions: Riboflavin-mediated PDT shows promise as a safe adjunctive therapy for periodontal infections. Larger, well-designed clinical trials with standardized parameters and extended follow-up are needed to further evaluate its efficacy and optimize treatment protocols for routine clinical application. Full article
(This article belongs to the Special Issue Natural Products in Photodynamic Therapy)
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<p>A Schematic representation of clinical and chemical aspects of riboflavin-mediated PDT [<a href="#B4-pharmaceutics-17-00217" class="html-bibr">4</a>].</p>
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<p>Prisma 2020 flow diagram.</p>
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15 pages, 3595 KiB  
Article
Enhancement of In Vivo Bone Regeneration by the Carbohydrate Derivative DP2
by Nissrine Ballout, Sylvestre Toumieux, Walaa Darwiche, Cathy Gomila, Eric Trécherel, Franck Accadbled, Sara Laurencin-Dalicieux, Isabelle Gennero, José Kovensky, Agnès Boullier and Jérôme Ausseil
Pharmaceuticals 2025, 18(2), 215; https://doi.org/10.3390/ph18020215 - 5 Feb 2025
Viewed by 572
Abstract
Background/Objectives: Delays in bone healing and complications of remodeling constitute a major medical problem—particularly in older adults and patients with comorbidities. Current therapeutic approaches are based on strategies that promote bone regeneration. We recently identified a disaccharide compound (DP2) that enhances in [...] Read more.
Background/Objectives: Delays in bone healing and complications of remodeling constitute a major medical problem—particularly in older adults and patients with comorbidities. Current therapeutic approaches are based on strategies that promote bone regeneration. We recently identified a disaccharide compound (DP2) that enhances in vitro mineralization in human osteoblast cells via the early activation of Runx2 and the induction of osteoblast differentiation. Methods: First, a calcium quantification assay was performed to assess mineralization in MC3T3-E1 cells. Next, microcomputed tomography and histological analyses were used to examine in vivo bone repair in a rat 5 mm cranial defect model following the implantation of DP2 coupled to a micro/macroporous biphasic CaP ceramic (MBCP+) or collagen scaffold. Results: Here, we demonstrated that DP2 induced osteogenic differentiation and significantly elevated calcium matrix deposition in the murine preosteoblast cell line MC3T3-E1. We found that treatment with DP2 coupled to MBCP+ repaired the calvarial defect on post-implantation day 91. It significantly increased bone mineral density starting on day 29 post-treatment. In addition, DP2 did not induce ectopic bone formation. Conclusions: Taken as a whole, these results show that DP2 is a promising candidate treatment for delayed bone healing. Full article
(This article belongs to the Section Pharmacology)
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<p><b>Effect of DP2 on the mineralization of MC3T3-E1 cells</b> MC3T3-E1 cells were incubated with either 10 mM β-gp, 10 mM β-gp + 30 μM DP2, or 10 mM β-gp + 100 ng/mL BMP-2 for 25 days. Intracellular calcium was quantified using the OCP colorimetric method and normalized as a percentage of the value for cells treated with β-gp alone 100 (n = 4). Data are expressed as the mean ± SEM. The <span class="html-italic">p</span>-value was determined using a one-way ANOVA, followed by Tukey’s test for multiple comparisons (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, ns: non-significant).</p>
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<p><b>Bone mineralization density (BMD, g/cm<sup>3</sup>) after implantation.</b> (<b>A</b>,<b>B</b>) Four groups of eight animals each were tested: MBCP<sup>+</sup> alone, MBCP<sup>+</sup> + 14 µg DP2, MBCP<sup>+</sup> + 42 µg DP2, and MBCP<sup>+</sup> + 10 µg BMP-2. BMD was calculated using CTAn software (version 1.14.4.1). Data are expressed as the mean ± SEM (n = 8). The <span class="html-italic">p</span>-value was determined in a one-way ANOVA, followed by Tukey’s test for multiple comparisons (*** <span class="html-italic">p</span> &lt; 0.001 for the empty defect vs. all the other groups; <span>$</span> <span class="html-italic">p</span> &lt; 0.01 for MBCP<sup>+</sup> alone vs. MBCP<sup>+</sup> + 14 µg DP2; # <span class="html-italic">p</span> &lt; 0.05 for MBCP<sup>+</sup> alone vs. MBCP<sup>+</sup> + 42 µg DP2) The table summarizes the results obtained for each group (g/cm<sup>3</sup>). (<b>C</b>,<b>D</b>) Four groups of eight animals each were tested: collagen alone, collagen + 14 µg DP2, collagen + 42 µg DP2, and collagen + 10 µg BMP-2. BMD was calculated using CTAn software. Data were expressed as the mean ± SEM (n = 8). The <span class="html-italic">p</span>-value was determined in a one-way ANOVA, followed by Tukey’s test for multiple comparisons (* <span class="html-italic">p</span> &lt; 0.05 for the empty defect vs. collagen + 14 µg DP2 and for the empty defect vs. collagen + 42 µg DP2). The table summarizes the obtained results for each group (g/cm<sup>3</sup>).</p>
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<p><b>Bone volume (BV/TV, %) after implantation.</b> (<b>A</b>) Four groups of eight animals were tested: MBCP<sup>+</sup> alone, MBCP<sup>+</sup> + 14 µg DP2, MBCP<sup>+</sup> + 42 µg DP2, and MBCP<sup>+</sup> + 10 µg BMP-2. (<b>B</b>) Four groups of eight animals were tested: collagen alone, collagen + 14 µg DP2, collagen + 42 µg DP2, and collagen + 10 µg BMP-2. BV/TV was calculated using CTAn software. Data were expressed as the mean ± SEM (n = 8). The <span class="html-italic">p</span>-value was determined using one-way ANOVA, followed by Tukey’s test for multiple comparisons (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p>
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<p><b>Micro-CT images of calvarial defects on post-implantation day 91.</b> A coronal view (<b>left image</b>) and sagittal view (<b>right image</b>) of the defect are shown for each condition, and the images are representative of the animals in each group ((<b>A</b>–<b>F</b>): Empty defect, (<b>B</b>–<b>G</b>): scaffold alone, (<b>C</b>–<b>H</b>): scaffold + 14 µg DP2, (<b>D</b>–<b>I</b>): scaffold + 42 µg DP2, and (<b>E</b>–<b>J</b>): scaffold + 10 µg BMP-2). The blue rectangle and circle indicate the region of interest, the blue arrows represent ectopic bone, and the white arrows represent empty spaces between the original bone and the newly formed bone.</p>
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<p><b>Histologic assessments of calvarial defects (MBCP<sup>+</sup> scaffold) on post-implantation day 91.</b> Bone sections were stained with H&amp;E reagent. Black arrow: newly formed bone; black asterisk: residual MBCP<sup>+</sup> scaffold. The blue rectangle represents the region of interest, and the red square represents the high-magnification insets E to H. (<b>A</b>–<b>E</b>) MBCP<sup>+</sup> alone, (<b>B</b>–<b>F</b>) MBCP<sup>+</sup> + 14 µg DP2, (<b>C</b>–<b>G</b>) MBCP<sup>+</sup> + 42 µg DP2, and (<b>D</b>–<b>H</b>) MBCP<sup>+</sup> + 10 µg BMP-2. Scale bars = 500 µm (<b>A</b>–<b>D</b>) or 100 µm (<b>E</b>–<b>H</b>).</p>
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<p><b>Histologic assessments of calvarial defects (collagen scaffold) on post-implantation day 91.</b> Bone sections were stained with H&amp;E reagent. Black arrow: newly formed bone; black asterisk: residual collagen matrix; the blue rectangle represents the region of interest; the red square represents the region the high-magnification insets E to H. (<b>A</b>–<b>E</b>) collagen alone, (<b>B</b>–<b>F</b>) collagen + 14 µg DP2, (<b>C</b>–<b>G</b>) collagen + 42 µg DP2, (<b>D</b>–<b>H</b>) and collagen + 10 µg BMP-2. Scale bars = 500 µm (<b>A</b>–<b>D</b>) or 100 µm (<b>E</b>–<b>H</b>).</p>
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<p><b>Synthesis and structure of DP2.</b> The acceptor glucoside was obtained in eight steps, with total control over the alpha stereochemistry of the aglycone moiety. The activated alpha trichloroacetimidate donor glucoside holding a 2-aminotrichloroacetyl group was obtained in a straightforward, three-step sequence [<a href="#B24-pharmaceuticals-18-00215" class="html-bibr">24</a>] and exclusively lead to the disaccharide with a beta configuration.</p>
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<p><b>The experimental protocol.</b> Two 5 mm diameter cranial lesions were created on each side of the sagittal suture using a diamond bur. Eight groups of animals (n = 8 each) were treated with (i) MBCP+ alone, (ii) MBCP<sup>+</sup> + 14 µg DP2, (iii) MBCP<sup>+</sup> + 42 µg DP2, (iv) MBCP<sup>+</sup> + 10 µg BMP-2, (v) collagen alone, (vi) collagen + 14 µg DP2, (vii) collagen + 42 µg DP2, or (viii) collagen + 10 µg BMP-2. Each scaffold was implanted on the left side; the right side remained empty and served as an internal control. A µCT scan was performed on post-implantation days 14, 29, 63, and 91. On post-implantation day 91, the rats were euthanized with CO2. The region of interest comprising the two calvarial defects was then harvested for histological analysis.</p>
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13 pages, 2031 KiB  
Article
Exposure of Xenogeneic Biomaterial to the Oral Environment and Its Impact on Tissue Healing of Immediate Dental Implants: A Case–Control Study
by Valessa F. Carvalho, João Garcez-Filho, Roberta Okamoto, Paula B. Frigério, Priscila L. Santos, Arthur B. Novaes Junior, Michel R. Messora and Mario Taba Jr
Appl. Sci. 2025, 15(2), 993; https://doi.org/10.3390/app15020993 - 20 Jan 2025
Viewed by 389
Abstract
This study evaluated the clinical and tomographic outcomes of socket healing. Immediate implants were placed in the molar area, and the gap was filled with either deproteinized bovine bone mineral (B) or collagen matrix (BM), n = 14/group. Scores of epithelization healing, immunoassay [...] Read more.
This study evaluated the clinical and tomographic outcomes of socket healing. Immediate implants were placed in the molar area, and the gap was filled with either deproteinized bovine bone mineral (B) or collagen matrix (BM), n = 14/group. Scores of epithelization healing, immunoassay for VEGF, IL-1β, and FGF from wound exudate, keratinized mucosa variation (ΔKM), and bone levels were evaluated. The B group had slower tissue maturation than BM (p < 0.05), but gingival epithelialization was similar (p > 0.05). At the restorative phase, the B group exhibited greater ΔKM at prosthesis installation—1 to 2 months of postoperative (increase of 0.29 mm) compared to the BM group (reduction of −1.5 mm) (p < 0.05). Inflammatory tissue responses as well as vertical and horizontal bone remodeling were similar (p > 0.05). Crestal bone remodeling was limited to less than 0.8 mm for both groups. Taken together, the B and BM groups behaved similarly and promoted stable conditions for biomaterial incorporation in the socket healing after immediate implant placement in molar areas. Full article
(This article belongs to the Section Applied Dentistry and Oral Sciences)
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<p>(<b>a</b>) Illustrative diagram of horizontal bone thickness measurements from the reference line (RL); (<b>b</b>) vertical soft tissue measurement; (<b>c</b>) horizontal reference lines and superimposition of the DICOM (Digital Imaging and Communications in Medicine) of the two CBCT scans on T1 and T2; (<b>d</b>) vertical reference lines at the lingual and buccal bone wall.</p>
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<p>Changes in the width of keratinized mucosa expressed in mm (n = 28). ∆ represents the net change in keratinized mucosa at different evaluation times: preoperative (baseline), after 1–2 months post-implant placement, and after the emergence contour of a provisional prosthesis. * indicate <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Wound healing phases in sequence: (<b>a</b>,<b>f</b>) before tooth extraction, (<b>b</b>,<b>g</b>) immediately after implant placement, (<b>c</b>,<b>h</b>) 2 days postoperatively (PO), (<b>d</b>,<b>i</b>) 7 days PO, and (<b>e</b>,<b>j</b>) 30 days PO. a-e represents the Bio-Oss + Mucograft treatment group (BM), while (<b>f</b>–<b>j</b>) represents the Bio-Oss treatment group (B).</p>
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<p>Levels of cytokines and growth factors. Molecules were measured by a Luminex assay in patients after implant placement. (<b>a</b>) FGF-2; (<b>b</b>) VEGF; (<b>c</b>) IL1-β. Abbreviation: B, Bio-Oss treatment; BM, Bio-Oss + Mucograft treatment; D: day after implant placement. * indicate <span class="html-italic">p</span> &lt; 0.05.</p>
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20 pages, 318 KiB  
Review
Narrative Review and Guide: State of the Art and Emerging Opportunities of Bioprinting in Tissue Regeneration and Medical Instrumentation
by Jaroslava Halper
Bioengineering 2025, 12(1), 71; https://doi.org/10.3390/bioengineering12010071 - 15 Jan 2025
Viewed by 524
Abstract
Three-dimensional printing was introduced in the 1980s, though bioprinting started developing a few years later. Today, 3D bioprinting is making inroads in medical fields, including the production of biomedical supplies intended for internal use, such as biodegradable staples. Medical bioprinting enables versatility and [...] Read more.
Three-dimensional printing was introduced in the 1980s, though bioprinting started developing a few years later. Today, 3D bioprinting is making inroads in medical fields, including the production of biomedical supplies intended for internal use, such as biodegradable staples. Medical bioprinting enables versatility and flexibility on demand and is able to modify and individualize production using several established printing methods. A great selection of biomaterials and bioinks is available, including natural, synthetic, and mixed options; they are biocompatible and non-toxic. Many bioinks are biodegradable and they accommodate cells so upon implantation, they integrate within the new environment. Bioprinting is suitable for printing tissues using living or viable components, such as collagen scaffolding, cartilage components, and cells, and also for printing parts of structures, such as teeth, using artificial man-made materials that will become embedded in vivo. Bioprinting is an integral part of tissue engineering and regenerative medicine. The addition of newly developed smart biomaterials capable of incorporating dynamic changes in shape depending on the nature of stimuli led to the addition of the fourth dimension of time in the form of changing shape to the three static dimensions. Four-dimensional bioprinting is already making significant inroads in tissue engineering and regenerative medicine, including new ways to create dynamic tissues. Its future lies in constructing partial or whole organ generation. Full article
(This article belongs to the Special Issue The New Frontiers of Artificial Organs Engineering)
17 pages, 4300 KiB  
Article
Histology Assessment of Chitosan–Polyvinyl Alcohol Scaffolds Incorporated with CaO Nanoparticles
by Carlos David Grande-Tovar, Jorge Ivan Castro Castro, Lemy Vanessa Barba-Rosado, Paula A. Zapata, Daniel Insuasty and Carlos-Humberto Valencia-Llano
Molecules 2025, 30(2), 276; https://doi.org/10.3390/molecules30020276 - 12 Jan 2025
Viewed by 567
Abstract
Scaffolds for regenerative therapy can be made from natural or synthetic polymers, each offering distinct benefits. Natural biopolymers like chitosan (CS) are biocompatible and biodegradable, supporting cell interactions, but lack mechanical strength. Synthetic polymers like polyvinyl alcohol (PVA) provide superior mechanical strength and [...] Read more.
Scaffolds for regenerative therapy can be made from natural or synthetic polymers, each offering distinct benefits. Natural biopolymers like chitosan (CS) are biocompatible and biodegradable, supporting cell interactions, but lack mechanical strength. Synthetic polymers like polyvinyl alcohol (PVA) provide superior mechanical strength and cost efficiency but are not biodegradable or supportive of cell adhesion. Combining these polymers optimizes their advantages while adding metal oxide nanoparticles like calcium oxide (CaO NPs) enhances antimicrobial properties by damaging bacterial membranes. In this study, we obtained the formation of CaO NPs by calcinating eggshells, which were mixed in a polymeric network of CS and PVA to obtain four different membrane formulations for subdermal tissue regeneration. The spherical nanoparticles measured 13.43 ± 0.46 nm in size. Their incorporation into the membranes broadened the hydroxyl bands in the Fourier transform infrared (FTIR) analysis at 3331 cm⁻1. X-ray diffraction (XRD) analysis showed changes in the crystalline structure, with new diffraction peaks at 2θ values of 7.2° for formulations F2, F3, and F4, likely due to the increased amorphous nature and concentration of CaO NPs. Additionally, higher CaO NPs concentrations led to a reduction in thermal properties and crystallinity. Scanning electron microscopy (SEM) revealed a heterogeneous morphology with needle-like structures on the surface, resulting from the uniform dispersion of CaO NPs among the polymer chains and the solvent evaporation process. A histological examination of the implanted membranes after 60 days indicated their biocompatibility and biodegradability, facilitated by incorporating CaO NPs. During the degradation process, the material fragmented and was absorbed by inflammatory cells, which promoted the proliferation of collagen fibers and blood vessels. These findings highlight the potential of incorporating CaO NPs in soft tissue regeneration scaffolds. Full article
(This article belongs to the Section Materials Chemistry)
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<p>(<b>A</b>) TEM image, (<b>B</b>) histogram of CaO nanoparticles, (<b>C</b>) XRD pattern, and (<b>D</b>) FTIR spectra of CaO nanoparticles obtained from eggshells.</p>
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<p>FTIR spectrum of PVA/CS/CaO NP membranes. F1, 30% CS/70% PVA; F2, 28% CS/70% PVA/2% CaO-NPs; F3, 26% CS/70% PVA/4% CaO-NPs; F4, 24% PCL/70% PLA/6% CaO-NPs.</p>
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<p>XRD diffractogram of CS/PVA/CaO NP membranes. F1, 30% CS/70% PVA; F2, 28% CS/70% PVA/2% CaO NPs; F3, 26% CS/70% PVA/4% CaO NPs; F4, 24% CS/70% PVA/6% CaO NPs.</p>
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<p>Thermogram (<b>A</b>) and its derivative curves (<b>B</b>) of the CS/PVA/CaO NP membranes. F1, 30% CS/70% PVA; F2, 28% CS/70% PVA/2% CaO NPs; F3, 26% CS/70% PVA/4% CaO NPs; F4, 24% CS/70% PVA/6% CaO NPs.</p>
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<p>DSC thermograms of the CS/PVA/CaO NP membranes. F1, 30% CS/70% PVA; F2, 28% CS/70% PVA/2% CaO NPs; F3, 26% CS/70% PVA/4% CaO NPs; F4, 24% CS/70% PVA/6% CaO NPs.</p>
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<p>Morphology of PVA/CS/CaO NP membranes by SEM. F1, 30% CS/70% PVA at 1000× and 10,000×; F2, 28% CS/70% PVA/2% CaO NPs at 1000× and 10,000×; F3, 6% CS/70% PVA/4% CaO NPs at 1000× and 10,000×; F4, 24% PCL/70% PLA/6% CaO NPs at 1000× and 10,000×.</p>
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<p>Macroscopic image of the dorsal surface implantation area in Wistar rats. (<b>A</b>): Biomodel with dorsal hair. (<b>B</b>): Dorsal surface with trichotomy. (<b>C</b>): Dorsal surface, internal aspect. Black rectangle: Intervened area. IZ: Implantation area. White arrows: Sites where specimens were implanted.</p>
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<p>Samples were implanted subdermally for 30 days. <b>A</b>–<b>L</b> correspond to formulation F1; <b>D</b>–<b>F</b> correspond to formulation F2; <b>G</b>–<b>I</b> correspond to formulation F3 and <b>J</b>–<b>L</b> correspond to formulation F4. (<b>A</b>): 4× image, HE technique. (<b>B</b>): Image at 4×, GT technique. (<b>C</b>): Image at 40×, MT technique. (<b>D</b>): Image at 10×, HE technique. (<b>E</b>): 10× image, GT technique. (<b>F</b>): 100× image, MT technique. (<b>G</b>): 4× image, MT technique. (<b>H</b>): 4× image, HE technique. (<b>I</b>): 100× image, HE technique. (<b>J</b>): 4× image, HE technique. (<b>K</b>): 100× image, GT technique. (<b>L</b>): 4× image, Mt technique. White circle: magnetization zone. IZ: Implantation zone. Red star: Fibrous cap. Yellow star: Inflammatory infiltrate. White arrow: Implanted material. Blue arrow: type III collagen fibers. M: Muscle. H: Hypodermis. Red circle: area of interest where fragments of the material are being detached. Red arrow: inflammatory cells. Red oval: area of interest where the material is cracking. Green arrow: type I collagen.</p>
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<p>Methodology for the synthesis of CaO NPs and the preparation of CS/PVA/CaO NP membranes.</p>
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20 pages, 40344 KiB  
Article
From Cartilage to Matrix: Protocols for the Decellularization of Porcine Auricular Cartilage
by Ana Caroline dos Santos, Livia Maria Barbosa de Andrade, Raí André Querino Candelária, Juliana Casanovas de Carvalho, Maria Carolina Miglino Valbão, Rodrigo da Silva Nunes Barreto, Marcelo Domingues de Faria, Rogerio Leone Buchaim, Daniela Vieira Buchaim and Maria Angelica Miglino
Bioengineering 2025, 12(1), 52; https://doi.org/10.3390/bioengineering12010052 - 9 Jan 2025
Viewed by 476
Abstract
The shortage of tissues and damaged organs led to the development of tissue engineering. Biological scaffolds, created from the extracellular matrix (ECM) of organs and tissues, have emerged as a promising solution for transplants. The ECM of decellularized auricular cartilage is a potential [...] Read more.
The shortage of tissues and damaged organs led to the development of tissue engineering. Biological scaffolds, created from the extracellular matrix (ECM) of organs and tissues, have emerged as a promising solution for transplants. The ECM of decellularized auricular cartilage is a potential tool for producing ideal scaffolds for the recellularization and implantation of new tissue in damaged areas. In order to be classified as an ideal scaffold, it must be acellular, preserving its proteins and physical characteristics necessary for cell adhesion. This study aimed to develop a decellularization protocol for pig ear cartilage and evaluate the integrity of the ECM. Four tests were performed using different methods and protocols, with four pig ears from which the skin and subcutaneous tissue were removed, leaving only the cartilage. The most efficient protocol was the combination of trypsin with a sodium hydroxide solution (0.2 N) and SDS (1%) without altering the ECM conformation or the collagen architecture. In conclusion, it was observed that auricular cartilage is difficult to decellularize, influenced by material size, exposure time, and the composition of the solution. Freezing and thawing did not affect the procedure. The sample thickness significantly impacted the decellularization time. Full article
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<p>Experimental design. In Protocol 1 (Prot. 1), the dissected sample was cut into 1 cm<sup>2</sup> fragments and agitated at room temperature for 10 days, followed by a vacuum pump for 2 h on days 9 and 10. Histological analyses were conducted using hematoxylin/eosin (H&amp;E) and DAPI staining. In Protocol 2 (Prot. 2), the sample was cut into 8 mm<sup>2</sup> fragments. Group A was frozen at −150 °C, while Group B was frozen at −80 °C, with both groups proceeding to agitation for 28 days. Histological analyses were also conducted using H&amp;E and DAPI staining. In Protocol 3 (Prot. 3), the sample was again cut into 8 mm<sup>2</sup> fragments, with one group frozen in liquid nitrogen and the other left unfrozen. Both groups were subjected to orbital agitation before histological analysis using H&amp;E and DAPI staining. In Protocol 4 (Prot. 4), the samples were cut into 1 cm<sup>2</sup> fragments and subjected to orbital agitation for decellularization and subsequent washings.</p>
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<p>Histological analyses of porcine ears subjected to Protocol 1 after 10 days of decellularization. Group (<b>A</b>) was treated with 1% SDS. Group (<b>B</b>) was treated with 0.5% SDS + 0.5% Triton X-100. Both groups were stained with H&amp;E and subjected to DAPI immunofluorescence. Scale bar: 200 μm. The white arrows indicate the cytoplasm and cellular nuclei highlighted by H&amp;E staining (<b>B</b>,<b>C</b>) and DAPI immunofluorescence (<b>D</b>–<b>F</b>).</p>
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<p>Histological sections of cartilage stained with H&amp;E and DAPI from Pilot 2 at 8, 17, and 28 days of decellularization from protocols with the combination of two detergents. Scale bar: 200 μm.</p>
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<p>Histological sections of cartilage stained with H&amp;E and DAPI from Pilot 2 at 8, 17, and 28 days of decellularization, using only one detergent. Scale bar: 200 μm.</p>
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<p>Histological sections of decellularized porcine auricular cartilage tissue stained with hematoxylin and eosin (H&amp;E) in Protocol 3. Scale bar: 200 μm. Group A (<b>A</b>–<b>D</b>) was not subjected to liquid nitrogen freezing, whereas Group B was (<b>E</b>–<b>H</b>). Blue arrows indicate areas devoid of chondrocytes, and black squares indicate damage to the extracellular matrix architecture caused by nitrogen and trypsin.</p>
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<p>DAPI fluorescence imaging of decellularized porcine auricular cartilage tissue from Protocol 3. Group A (<b>A</b>–<b>D</b>) was not subjected to liquid nitrogen freezing, whereas Group B was (<b>E</b>–<b>H</b>). Scale bar: 200 μm.</p>
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<p>Histological sections of decellularized porcine auricular cartilage tissue stained with Gomori’s trichrome from Protocol 3. Group A (<b>A</b>–<b>D</b>) was not subjected to liquid nitrogen freezing, whereas Group B was (<b>E</b>–<b>H</b>). Scale bar: 200 μm. Black arrows highlight areas with high collagen abundance, even after the decellularization process.</p>
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<p>Histological sections of decellularized porcine auricular cartilage tissue stained with Alcian Blue from Protocol 3. Group A (<b>A</b>–<b>D</b>) was not subjected to liquid nitrogen freezing, whereas Group B was (<b>E</b>–<b>H</b>). Scale bar: 200 μm.</p>
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<p>Histological sections of decellularized porcine auricular cartilaginous tissue stained with safranin O from Protocol 3. Scale bar: 200 μm.</p>
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<p>Histological analysis and scanning electron microscopy of porcine auricular cartilage samples submitted to Protocol 4. Scale bar: 100 μm. (<b>A</b>–<b>C</b>) Control group. (<b>D</b>–<b>F</b>) Decellularized tissue. Blue arrows indicate the presence of nuclei in the control group and intact cellular lacunae, whereas black arrows highlight the absence of nuclei after decellularization, whereas yellow arrows highlight empty lacunae and the absence of cell bodies.</p>
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<p>Photomicrograph of porcine auricular cartilage stained with Gomori’s trichrome, alcian blue, safranin O and picrosirius red (Protocol 4). Scale bar: 100 μm. Decellularized group (<b>E</b>–<b>H</b>) compared to the control group (<b>A</b>–<b>D</b>).</p>
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<p>Immunohistochemical analysis of porcine auricular cartilage stained for elastin, laminin, fibronectin, collagen I, collagen III, and collagen V. Red arrows indicate regions with positive expression of the matrix proteins elastin, collagen I, collagen III, and collagen V. Scale bar: 100 μm.</p>
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<p>Quantification of glycosaminoglycans (GAGs) (<b>A</b>) and quantification of total collagen (<b>B</b>) in native (control) and decellularized porcine auricular cartilage tissue. Results are expressed as micrograms per milligram of tissue (µg/mg).</p>
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14 pages, 1169 KiB  
Article
Antimicrobial Efficacy of Five Wound Irrigation Solutions in the Periprosthetic Joint Infection Microenvironment In Vitro and Ex Vivo
by Anja L. Honegger, Tiziano A. Schweizer, Yvonne Achermann and Philipp P. Bosshard
Antibiotics 2025, 14(1), 25; https://doi.org/10.3390/antibiotics14010025 - 3 Jan 2025
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Abstract
Background/Objectives: Periprosthetic joint infections (PJI) are difficult to treat due to biofilm formation on implant surfaces and the surrounding tissue, often requiring removal or exchange of prostheses along with long-lasting antibiotic treatment. Antiseptic irrigation during revision surgery might decrease bacterial biofilm load and [...] Read more.
Background/Objectives: Periprosthetic joint infections (PJI) are difficult to treat due to biofilm formation on implant surfaces and the surrounding tissue, often requiring removal or exchange of prostheses along with long-lasting antibiotic treatment. Antiseptic irrigation during revision surgery might decrease bacterial biofilm load and thereby improve treatment success. This in vitro study investigated and compared the effect of five advanced wound irrigation solutions to reduce bacterial burden in the PJI microenvironment. Methods: We treated in vitro biofilms grown on titanium alloy implant discs with clinical bacterial strains isolated from patients with PJIs, as well as abscess communities in a plasma-supplemented collagen matrix. The biofilms were exposed for 1 min to the following wound irrigation solutions: Preventia®, Prontosan®, Granudacyn®, ActiMaris® forte (‘Actimaris’), and Octenilin®. We measured the bacterial reduction of these irrigation solutions compared to Ringer–Lactate and to the strong bactericidal but not approved Betaseptic solution. Additionally, ex vivo free-floating bacteria isolated directly from clinical sonication fluids were treated in the same way, and regrowth or lack of regrowth was recorded as the outcome. Results: Irrigation solutions demonstrated variable efficacy. The mean CFU log10 reduction was as follows: Octenilin, 3.07, Preventia, 1.17, Actimaris, 1.11, Prontosan, 1.03, and Granudacyn, 0.61. For SACs, the reduction was: Actimaris, 8.27, Octenilin, 0.58, Prontosan, 0.56, Preventia, 0.35, and Granudacyn, 0.24. Conclusions: All solutions achieved complete bacterial eradication in all tested ex vivo sonication fluids, except Granudacyn, which was ineffective in 33% of the samples (2 out of 6). Advanced wound irrigation solutions have the potential to reduce bacterial burden in the PJI microenvironment during revision surgery. However, their efficacy varies depending on bacterial species, growth state, and the composition of the irrigation solution. This underscores the importance of considering these factors when developing future PJI-specific irrigation solutions. Full article
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Figure 1

Figure 1
<p>Antimicrobial efficacy of the irrigation solutions on biofilms formed on titanium alloy (TAV) discs. (<b>A</b>) Log<sub>10</sub> CFUs/mL reduction of the irrigation solutions on 6-day-old <span class="html-italic">S. aureus</span>, <span class="html-italic">S. epidermidis</span>, and <span class="html-italic">E. coli</span>, as well as 8-day-old <span class="html-italic">C. acnes</span> biofilms formed on TAV discs. Results are depicted as a relative reduction to Ringer’s lactate solution (negative control) and are the mean (±SEM) of three independent experiments performed in duplicates. Betaseptic (red) is not a wound irrigation solution but was used as a positive control with strong bactericidal activity. (<b>B</b>) Heat map indicating the relative log<sub>10</sub> CFUs/mL reductions across all irrigation solutions and bacterial species. (<b>C</b>) Heat map indicating the mean log<sub>10</sub> CFUs/mL reduction of each irrigation solution across all bacteria. Statistical analyses in A and C were performed by two-way ANOVA. Statistically significant reductions are indicated with asterisks: *, <span class="html-italic">p</span> &lt; 0.5; **, <span class="html-italic">p</span> &lt; 0.1; ****, <span class="html-italic">p</span> &lt; 0.0001.</p>
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<p>Antimicrobial efficacy of the irrigation solutions in the <span class="html-italic">S. aureus</span> abscess communities (SAC) model. (<b>A</b>) Representative image of SACs grown in collagen. (<b>B</b>) Log<sub>10</sub> CFUs/mL reduction of the irrigation solutions on 24 h old SACs formed in plasma-supplemented collagen matrices. Results are depicted as a relative reduction to Ringer’s lactate solution (negative control) and are the mean (±SEM) of three independent experiments performed in duplicates. Betaseptic (red) is not a wound irrigation solution. Statistically significant reductions are indicated with asterisks: ****, <span class="html-italic">p</span> &lt; 0.0001.</p>
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