In Vitro Evaluation of Mangostanin as an Antimicrobial and Biocompatible Topical Antiseptic for Skin and Oral Tissues.

Skin and oral tissue infections pose significant health challenges worldwide, necessitating the exploration of new antiseptic agents that are both effective and biocompatible. This study evaluated the antibacterial efficacy and biocompatibility of mangostanin (MGTN), a xanthone derived from Garcinia mangostana L., against commercial antiseptics across various bacterial strains (Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Cutibacterium acnes) and in vitro models of skin and oral tissues. MGTN demonstrated significant antimicrobial activity against all tested pathogens concurrently exhibiting negligible cytotoxic effects on human gingival fibroblasts as well as on three-dimensional (3D) models of human epidermis and oral epithelium. Furthermore, using pooled human saliva, MGTN effectively inhibited plaque biofilm formation, suggesting its potential as a natural, biocompatible antiseptic for skin and oral health applications. These findings position MGTN as a promising candidate for further development into antiseptic formulations, offering a natural alternative to current synthetic options.

Intra-articular injection of platelet lysate-derived extracellular vesicles recovers from knee osteoarthritis in an in vivo rat model.

Objective: MSCs and Platelet-Rich Plasma are the main focus in the study of new regenerative treatments aimed to reverse Osteoarthritis (OA). However, extracellular vesicles (EVs) present several advantages to cell-based treatments. Thus, the aim of this study was to compare and evaluate the regenerative potential of MSC-derived EVs (cEVs) and platelet-derived EVs (pEVs) in an OA cartilage rat model. Design: OA in vivo model was established through injection of 6 mg MIA in the rat knee joints. After 14 and 21 days, OA knee joints were treated with 1 × 1010 particles of pEVs or cEVs. At day 28, the animals were sacrificed, plasma was collected to quantify CTX-II and knee joints were excised to be evaluated by Cone Beam Computed Tomography (CBCT). After decalcification, histology was used to determine the OARSI score and to visualize collagen and glycosaminoglycan content. Results: pEVs and cEVs samples did not show significant differences per se but they did in terms of regenerative effects on OA knee joints. pEVs-treated knee joints showed better subchondral bone integrity in CT-analysed parameters when compared to cEVs or OA group, showing similar values to the healthy control group. Moreover, OARSI score indicated that pEVs showed a greater OA reversion in knee joints, especially in female rats, and so indicated the analysed histological images. Conclusions: pEVs are proposed as a viable regeneration treatment for OA since they are not only capable of exerting their regenerative potential on osteoarthritic cartilage, but also outperform cEVs in terms of efficacy, particularly in females. Significance statement: Osteoarthritis (OA) is one of the most age-related diseases. It is estimated that 500 million people suffer from OA worldwide, representing the principal cause of chronic disability in adults. In the present study we evaluated the therapeutic effect of extracellular vesicles (EVs) from different sources (platelet lysate and human umbilical cord mesenchymal stromal cells) in an in vivo rat model. Our results demonstrate that platelet-derived EVs (pEVs) induce an OA reversion in knee joints, thus evidencing the therapeutic potential of pEVs as cell-free regenerative agents for OA treatment. The translational potential of this article: Platelet-derived extracellular vesicles (pEVs) offer a promising cell-free therapy option for OA treatment. Their production could be easily standardized and reproduced without extensive platelet harvesting and amplification, thus paving the way for their clinical translation.

Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model.

Aims: Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. Methods: pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 109 particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs. Results: Significantly higher content of DNA and collagen was observed for the pEV-treated group compared to control and cEV groups. No differences were found in GAG quantification nor in EVs uptake within any treated group. Conclusion: In conclusion, pEVs showed better performance than cEVs in our in vitro OA model. Although further studies are needed, pEVs are shown as a potential alternative to cEVs for cell-free regenerative medicine.

A Sequential Micro-Immunotherapy Medicine Increases Collagen Deposition in Human Gingival Fibroblasts and in an Engineered 3D Gingival Model under Inflammatory Conditions.

Periodontal therapies use immune mediators, but their side effects can increase with dosage. Micro-immunotherapy (MI) is a promising alternative that employs immune regulators at low and ultralow doses to minimize adverse effects. In this study, the effects of 5 capsules and the entire 10-capsule sequence of the sequential MI medicine (MIM-seq) were tested in two in vitro models of periodontitis. Firstly, human gingival fibroblasts (hGFs) exposed to interleukin (IL)-1ß to induce inflammation were treated with five different capsules of MIM-seq for 3 days or with MIM-seq for 24 days. Subsequently, MIM-seq was analyzed in a 3D model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus. Simultaneously, a non-IL-1ß-treated control and a vehicle were included. The effects of the treatments on cytotoxicity, collagen deposition, and the secreted levels of IL-1α, IL-6, prostaglandin E2 (PGE2), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) were evaluated. None of the tested items were cytotoxic. The complete sequence of MIM-seq decreased PGE2 release and restored collagen deposition levels induced by IL-1ß treatment in hGFs exposed to IL-1ß. MIM-seq treatment restored collagen production levels in both models. These promising preclinical findings suggest that MIM-seq should be further investigated for periodontitis treatment.

Platelet-derived extracellular vesicles formulated with hyaluronic acid gels for application at the bone-implant interface: An animal study.

Background/Objective: Platelet derived extracellular vesicles (pEV) are promising therapeutical tools for bone healing applications. In fact, several in vitro studies have already demonstrated the efficacy of Extracellular Vesicles (EV) in promoting bone regeneration and repair in various orthopedic models. Therefore, to evaluate the translational potential in this field, an in vivo study was performed. Methods: Here, we used hyaluronic acid (HA) gels formulated with pEVs, as a way to directly apply pEVs and retain them at the bone defect. In this study, pEVs were isolated from Platelet Lysate (PL) through size exclusion chromatography and used to formulate 2% HA gels. Then, the gels were locally applied on the tibia cortical bone defect of New Zeland White rabbits before the surgical implantation of coin-shaped titanium implants. After eight weeks, the bone healing process was analyzed through biomechanical, micro-CT, histological and biochemical analysis. Results: Although no biomechanical differences were observed between pEV formulated gels and non-formulated gels, biochemical markers of the wound fluid at the interface presented a decrease in Lactate dehydrogenase (LDH) activity and alkaline phosphatase (ALP) activity for pEV HA treated implants. Moreover, histological analyses showed that none of the treatments induced an irritative effect and, a decrease in the fibrotic response surrounding the implant for pEV HA treated implants was described. Conclusion: In conclusion, pEVs improve titanium implants biocompatibility at the bone-implant interface, decreasing the necrotic effects of the surgery and diminishing the fibrotic layer associated to the implant encapsulation that can lead to implant failure.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model.

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1ß, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1ß levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Extracellular Vesicle-Based Hydrogels for Wound Healing Applications.

Hydrogels and extracellular vesicle-based therapies have been proposed as emerging therapeutic assets in wound closure. The combination of these elements has given good results in managing chronic and acute wounds. The intrinsic characteristics of the hydrogels in which the extracellular vesicles (EVs) are loaded allow for overcoming barriers, such as the sustained and controlled release of EVs and the maintenance of the pH for their conservation. In addition, EVs can be obtained from different sources and through several isolation methods. However, some barriers must be overcome to transfer this type of therapy to the clinic, for example, the production of hydrogels containing functional EVs and identifying long-term storage conditions for EVs. The aim of this review is to describe the reported EV-based hydrogel combinations, along with the obtained results, and analyze future perspectives.

Synthesis and Modification of Gelatin Methacryloyl (GelMA) with Antibacterial Quaternary Groups and Its Potential for Periodontal Applications.

Gelatin methacryloyl (GelMA) hydrogels have been widely used for different biomedical applications due to their tunable physical characteristics and appropriate biological properties. In addition, GelMA could be modified with the addition of functional groups providing inherent antibacterial capabilities. Here, GelMA-based hydrogels were developed through the combination of a GelMA unmodified and modified polymer with quaternary ammonium groups (GelMAQ). The GelMAQ was synthesized from GelMA with a low degree of substitution of methacrylamide groups (DSMA) and grafted with glycidyltrimethylammonium chloride in the free amine groups of the lysine moieties present in the original gelatin. GelMAs with high DSMA and GelMAQ were combined 50/50% or 25/75% (w/w), respectively, and compared to controls GelMA and GelMA with added chlorhexidine (CHX) at 0.2%. The different hydrogels were characterized using 1H-NMR spectroscopy and swelling behavior and tested in (1) Porphyromonas gingivalis to evaluate their antibacterial properties and (2) human gingival fibroblast to evaluate their cell biocompatibility and regenerative properties. GelMA/GelMAQ 25/75% showed good antibacterial properties but also excellent biocompatibility and regenerative properties toward human fibroblasts in the wound healing assay. Taken together, these results suggest that the modification of GelMA with quaternary groups could facilitate periodontal tissue regeneration, with good biocompatibility and added antibacterial properties.

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications.

Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.

Customizing the extracellular vesicles release and effect by strategizing surface functionalization of titanium.

Metallic material functionalization with Extracellular Vesicles (EVs) is a desirable therapeutic approach to improve regenerative procedures. Among the different functionalization strategies available, here we have compared drop casting on machined Ti surfaces, drop casting on nanostructured TiO2 surfaces and polymeric entrapment with polydopamine. EVs are a heterogeneous population of communication nanovesicles released by cells that are being intensively investigated for their use in therapeutics. We have selected platelet derived EVs for Ti surface coating due to their demonstrated osteoinductive properties. Our results show that each functionalization strategy leads to differences in the size of EV populations attached to and released from the metallic implants, which, in turn, leads to variations in their osteogenic capability measured through alkaline phosphatase activity and calcium deposition. In conclusion, the functionalization strategy used has an important effect on the resulting implant functionality, probably due to the heterogeneous EVs nature. Thus, the methodological approach to metallic material functionalization should be carefully chosen when working with extracellular vesicles in order to obtain the desired therapeutic application.

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants.

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 µm sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Comparative In Vitro Evaluation of Commercial Periodontal Gels on Antibacterial, Biocompatibility and Wound Healing Ability.

In the last years, several studies testing commercial periodontal gels that contain chlorhexidine (CHX) or other antibacterial agents, have raised concerns regarding their cytotoxicity in periodontal tissues. We aimed at comparing the biocompatibility but also the efficacy as regards to the antibacterial and wound healing ability of different commercial periodontal gels. In vitro human gingival fibroblasts (GF) and a 3D model of human tissue equivalents of gingiva (GTE) were used under inflammatory conditions to evaluate wound closure, cytotoxicity and gene expression. Antibacterial effects were also investigated on Porphyromonas gingivalis growth, viability and gingipain activity. In GF and in the bacterial study, we found cytotoxic effects on GF and a high inhibition on bacterial growth rate in gels containing CHX, asiaticoside, enoxolone, cetylpyridinium chloride, propolis and eugenol. Of the two gels that were non-cytotoxic, Syntoss Biogel (containing chondrontin sulfate) and Emdogain (EMD, containing amelogenin and propylene glycol alginate), EMD showed the best wound closure, with no effect on P. gingivalis growth but decreased gingipain activity. On the other hand, Syntoss Biogel reduced viability and gingipain activity of P. gingivalis, but lack wound healing capacity. In the 3D GTE, Syntoss Biogel and EMD showed a good biocompatibility. Among all the tested gels, formulations containing CHX, asiaticoside, enoxolone, cetylpyridinium chloride, propolis and eugenol showed high antibacterial effect but also showed high cytotoxicity in eukaryotic cells. EMD was the one with the best biocompatibility and wound healing ability at the conditions tested.

Platelet-Derived Extracellular Vesicles for Regenerative Medicine.

Extracellular vesicles (EVs) present a great potential for the development of new treatments in the biomedical field. To be used as therapeutics, many different sources have been used for EVs obtention, while only a few studies have addressed the use of platelet-derived EVs (pEVs). In fact, pEVs have been shown to intervene in different healing responses, thus some studies have evaluated their regenerative capability in wound healing or hemorrhagic shock. Even more, pEVs have proven to induce cellular differentiation, enhancing musculoskeletal or neural regeneration. However, the obtention and characterization of pEVs is widely heterogeneous and differs from the recommendations of the International Society for Extracellular Vesicles. Therefore, in this review, we aim to present the main advances in the therapeutical use of pEVs in the regenerative medicine field while highlighting the isolation and characterization steps followed. The main goal of this review is to portray the studies performed in order to enhance the translation of the pEVs research into feasible therapeutical applications.

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants.

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as proteins and nucleic acids, which present great potential in regenerative medicine. More recently, EVs derived from Platelet Lysate (PL) have shown an osteogenic capability comparable to PL. Besides, biomaterials are frequently used in orthopedics or dental restoration. Here, we provide a method to functionalize Ti surfaces with PL-derived EVs in order to improve their osteogenic properties. EVs are isolated from PL by size exclusion chromatography, and afterward Ti surfaces are functionalized with PL-EVs by drop casting. Functionalization is proven by EVs release and its biocompatibility by the lactate dehydrogenase (LDH) release assay.

BMP4 micro-immunotherapy increases collagen deposition and reduces PGE2 release in human gingival fibroblasts and increases tissue viability of engineered 3D gingiva under inflammatory conditions.

BACKGROUND: We aimed to evaluate the effect of low doses (LD) bone morphogenetic protein-2 (BMP2) and BMP4 micro-immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration. METHODS: We first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1ß. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization-related enzymes (matrix metalloproteinase-1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long-term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release. RESULTS: LD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re-establish the impaired collagen metabolism induced by IL1ß treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days. CONCLUSIONS: LD BMP4 MI treatment shows anti-inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells.

AIMS: Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs). METHODS: EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca2+) content. RESULTS: Osteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response. CONCLUSION: PL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations.Cite this article: Bone Joint Res 2020;9(10):667-674.

Osteoimmunomodulatory Effects of Enamel Matrix Derivate and Strontium Coating Layers: A Short- and Long-Term In Vivo Study.

Over the past few years, surface modification of implant surfaces has gained substantial attention as a promising solution to avoid the failure of biomaterials after implantation. Although researchers suggest several strategies for surface functionalization of titanium-based implants, only a few studies have compared the osteoimmunomodulatory effects of ionic nanostructures and biofunctionalization in the same biological model. Enamel matrix derivate (EMD) and strontium are both known for their positive influences on bone cell responses. In this study, we functionalized the titanium-zirconium implant surface with EMD and strontium using an electrochemical cathodic polarization method. Afterward, we evaluated the osteoimmunomodulatory effects of EMD or strontium coated titanium-zirconium implants in the tibia of eight Gray Bastard Chinchilla rabbits. We performed 2 and 3D micro-CT, wound fluid, histologic, and histomorphometric analyses on bone tissues after 4- and 8-weeks of implantation. Although the results could indicate some differences between groups regarding the bone quality, there was no difference in bone amount or volume. EMD stimulated higher ALP activity and lower cytotoxicity in wound fluid, as well as a lower expression of inflammatory markers after 8 weeks indicating its osteoimmunomodulatory effects after implantation. Overall, the results suggested that ionic nanostructure modification and biofunctionalization might be useful in regulating the immune responses to implants.

Multifunctional Properties of Quercitrin-Coated Porous Ti-6Al-4V Implants for Orthopaedic Applications Assessed In Vitro.

(1) One strategy to improve the outcome of orthopedic implants is to use porous implants with the addition of a coating with an antibacterial biomolecule. In this study, we aimed to produce and test the biocompatibility, the osteopromotive (both under normal conditions and under a bacterial challenge with lipopolysaccharide (LPS)) and antibacterial activities of a porous Ti-6Al-4V implant coated with the flavonoid quercitrin in vitro. (2) Porous Ti-6Al-4V implants were produced by 3D printing and further functionalized with quercitrin by wet chemistry. Implants were characterized in terms of porosity and mechanical testing, and the coating with quercitrin by fluorescence staining. Implant biocompatibility and bioactivity was tested using MC3T3-E1 preosteoblasts by analyzing cytotoxicity, cell adhesion, osteocalcin production, and alkaline phosphatase (ALP) activity under control and under bacterial challenging conditions using lipopolysaccharide (LPS). Finally, the antibacterial properties of the implants were studied using Staphylococcus epidermidis by measuring bacterial viability and adhesion. (3) Porous implants showed pore size of about 500 µm and a porosity of 52%. The coating was homogeneous over all the 3D surface and did not alter the mechanical properties of the Young modulus. Quercitrin-coated implants showed higher biocompatibility, cell adhesion, and osteocalcin production compared with control implants. Moreover, higher ALP activity was observed for the quercitrin group under both normal and bacterial challenging conditions. Finally, S. epidermidis live/dead ratio and adhesion after 4 h of incubation was lower on quercitrin implants compared with the control. (4) Quercitrin-functionalized porous Ti-6Al-4V implants present a great potential as an orthopedic porous implant that decreases bacterial adhesion and viability while promoting bone cell growth and differentiation.