Análisis del cnidoma combinado con una evaluación in vitro del potencial lítico, cyto- y neurotóxicos de Cyanea Capillata (Cnidaria: Scyphozoa)

The cnidocysts and the lytic, cyto- and neurotoxic potency of a large specimen of Cyanea capillata (L.) with 55 cm umbrella diameter were compared with those of a pooled C. capillata sample (average ø 14 cm) in order to investigate organismal developments at a cellular and biochemical level. Nematocysts of the type A-isorhiza in both fishing tentacles and oral arms and the O-isorhizas of oral arms were enlarged in the 55 cm specimen. Additionally, the number of nematocysts per battery in the fishing tentacles was increased. Increased gill cell toxicity and neurotoxic activity were demonstrated for the fishing tentacle venom of the 55 cm C. capillata in comparison with the smaller medusae. A two-fold higher haemolytic activity was detected for the venom of oral arms obtained from the large C. capillata compared with the oral arm venom prepared from the smaller medusae.Con el fin de estudiar la evolución ontogenética a un nivel celular y bioquímico de los cnidocistos de la potencia lítica, y las características cito- y neurotóxicas de Cyanea capillata (L.), se ha comparado un ejemplar de 55 cm de diámetro de umbrela de la especie con una muestra de varios ejemplares de C. capillata (promedio 14 cm de diámetro). Tanto los nematocistos del tipo A isorhiza de los tentáculos marginales como en los mesentéricos y los nematocistos del tipo O isorhizas de tentáculos orales fueron mayores en el ejemplar de C. capillata de 55 cm. Además, el número de nematocistos por zona de agregación fue mayor en el ejemplar de 55 cm. En cuanto a la actividad tóxica de los cnidocistos, ésta fue superior tanto a nivel de célula como de la actividad de la toxina (veneno) en los tentáculos marginales del ejemplar de 55 cm frente a los ejemplares más pequeños de la especie. También se observó una actividad hemolítica de la toxina (veneno) dos veces superior en los cnidocistos de los tentáculos marginales del ejemplar grande, 55 cm de C. capillata frente a los ejemplares pequeños

Proteomic Analysis of Mesenchymal Stem Cells and Monocyte Co-Cultures Exposed to a Bioactive Silica-Based Sol–Gel Coating

New methodologies capable of extensively analyzing the cell-material interactions are necessary to improve current in vitro characterization methods, and proteomics is a viable alternative. Also, many studies are focused on monocultures, even though co-cultures model better the natural tissue. For instance, human mesenchymal stem cells (MSCs) modulate immune responses and promote bone repair through interaction with other cell types. Here, label-free liquid chromatography tandem mass spectroscopy proteomic methods were applied for the first time to characterize HUCPV (MSC) and CD14+ monocytes co-cultures exposed to a bioactive sol–gel coating (MT). PANTHER, DAVID, and STRING were employed for data integration. Fluorescence microscopy, enzyme-linked immunosorbent assay, and ALP activity were measured for further characterization. Regarding the HUCPV response, MT mainly affected cell adhesion by decreasing integrins, RHOC, and CAD13 expression. In contrast, MT augmented CD14+ cell areas and integrins, Rho family GTPases, actins, myosins, and 14-3-3 expression. Also, anti-inflammatory (APOE, LEG9, LEG3, and LEG1) and antioxidant (peroxiredoxins, GSTO1, GPX1, GSHR, CATA, and SODM) proteins were overexpressed. On co-cultures, collagens (CO5A1, CO3A1, CO6A1, CO6A2, CO1A2, CO1A1, and CO6A3), cell adhesion, and pro-inflammatory proteins were downregulated. Thus, cell adhesion appears to be mainly regulated by the material, while inflammation is impacted by both cellular cross-talk and the material. Altogether, we conclude that applied proteomic approaches show its potential in biomaterial characterization, even in complex systems.This work was supported by MINECO [MAT2017-86043-R; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091, BEFPI/2021/043, PROMETEO/2020/069], Universitat Jaume I [UJI-B2017-37], and the University of the Basque Country [GIU18/189]. Andreia Cerqueira was supported by the Margarita Salas postdoctoral contract MGS/2022/10 (UP2022-024) financed by the European Union-NextGenerationEU. The University Medical Centre Hamburg-Eppendorf (Hamburg, Germany) and the Clinics for Gynecology AGAPLESION BETHESDA Hospital provided the blood and tissue for cell isolation. The authors would like to thank Raquel Oliver, Jose Ortega, Iraide Escobés, and Anke Borkam-Schuster for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for producing the titanium discs

Proteomic Analysis of Mesenchymal Stem Cells and Monocyte Co-Cultures Exposed to a Bioactive Silica-Based Sol–Gel Coating

New methodologies capable of extensively analyzing the cell-material interactions are necessary to improve current in vitro characterization methods, and proteomics is a viable alternative. Also, many studies are focused on monocultures, even though co-cultures model better the natural tissue. For instance, human mesenchymal stem cells (MSCs) modulate immune responses and promote bone repair through interaction with other cell types. Here, label-free liquid chromatography tandem mass spectroscopy proteomic methods were applied for the first time to characterize HUCPV (MSC) and CD14+ monocytes co-cultures exposed to a bioactive sol–gel coating (MT). PANTHER, DAVID, and STRING were employed for data integration. Fluorescence microscopy, enzyme-linked immunosorbent assay, and ALP activity were measured for further characterization. Regarding the HUCPV response, MT mainly affected cell adhesion by decreasing integrins, RHOC, and CAD13 expression. In contrast, MT augmented CD14+ cell areas and integrins, Rho family GTPases, actins, myosins, and 14-3-3 expression. Also, anti-inflammatory (APOE, LEG9, LEG3, and LEG1) and antioxidant (peroxiredoxins, GSTO1, GPX1, GSHR, CATA, and SODM) proteins were overexpressed. On co-cultures, collagens (CO5A1, CO3A1, CO6A1, CO6A2, CO1A2, CO1A1, and CO6A3), cell adhesion, and pro-inflammatory proteins were downregulated. Thus, cell adhesion appears to be mainly regulated by the material, while inflammation is impacted by both cellular cross-talk and the material. Altogether, we conclude that applied proteomic approaches show its potential in biomaterial characterization, even in complex systems

In Vitro Investigation on Degradable Mg-Based Biomaterial under the Impact of the Serum Glycoprotein Fetuin

Biomedical applications of magnesium (Mg) and its alloys are generally dependent on their degradation behavior in vivo. Despite its attractive properties, which make Mg suitable for orthopedic applications, the in vivo material-tissue (bone, blood, and lymph tissues) interaction is not yet fully understood. To investigate the influence of major serum proteins on the degradation, this study focused on fetuin, which is one of the major non-collagenous plasma proteins and which is essential for biomineralization. This study used a physiological setup to investigate the influence of fetuin on the degradation behavior of pure Mg in the presence of calcium (Ca). Extruded pure Mg samples were immersed under cell culture conditions in Hank’s balanced salt solution (HBSS) under defined Ca regimes. The results showed a significant decrease in the degradation rate (DR) when both fetuin and Ca were present in an immersion medium as compared to media where they were not simultaneously present. A possible reason for this behavior was the forming of a dense, protein-degradation products protection barrier at the material surface. Furthermore, the limitation of freely available Ca might be a reason for a decreased degradation. The cultivation of primary osteoblasts (pOB) was possible at the fetuin-coated Mg-surface without additional serum supplementation

Preclinical in vivo research of magnesium-based implants for fracture treatment: A systematic review of animal model selection and study design

Background: Degradable magnesium implants are promising for clinical fracture treatment, providing less stress-shielding mechanical support and superior bone-strengthening benefits to traditional materials. The quality of preclinical research is essential for developing Mg implants; however, there are considerable variations in the model selection and study design in published studies, posing challenges for safe and effective clinical translation of lab discoveries. Objective: The purpose of this systematic review is to investigate the current progress on in vivo research of Mg implants for fracture treatment, focusing on model selection, Mg materials, implant design, methodological and analyzing techniques, aiming to provide comprehensive guidance for future preclinical research. Materials and methods: PubMed and Embase online databases were searched to identify researches investigating Mg implants in animal models of fracture from 1960 to December 2019, using a combination of keywords: magnesium and fracture. Eligible studies were included without language restriction. Data extraction was conducted for qualitative analysis. Meta-analysis was not performed due to extensive heterogeneities among studies. Results: Twenty studies published from 2014 to 2019 were included. Publication information, animal model, methodological quality, implants preparation, and evaluation techniques were extracted. The methodological assessment revealed low to high risk of bias among studies. Both rodent and non-rodent species were selected, and the anatomical sites for inducing fractures included both cranial-facial and limb bones. Pure Mg and alloys with or without surface modification were evaluated, covering implant designs of both intra-medullary and extra-medullary fixation. Radiological and histological evaluations were commonly conducted. Conclusions: Published in vivo evidence confirmed the role of Mg-based implants in promoting fracture healing. However, considering heterogeneity in animal selection, implant preparation, and evaluation methods, there still lacks a standardized reference model. By analyzing the information extracted from included studies, the systematic review may facilitate planning and conducting preclinical research with translational perspective

Peri‐implant gas accumulation in response to magnesium‐based musculoskeletal biomaterials: Reframing current evidence for preclinical research and clinical evaluation

Historically, the rapid degradation and massive gas release from magnesium (Mg) implants resulted in severe emphysema and mechanical failure. With the advent of new alloys and surface treatment methods, optimized Mg implants have re-entered clinics since last decade with reliable performance. However, the optimization aims at slowing down the degradation process, rather than exemption of the gas release. This study involved a systematic evaluation of current preclinical and clinical evidence, regarding the physical signs, symptoms, radiological features, pathological findings and complications potentially associated with peri‑implant gas accumulation (PIGA) after musculoskeletal Mg implantation. The literature search identified 196 potentially relevant publications, and 51 papers were enrolled for further analysis, including 22 preclinical tests and 29 clinical studies published from 2005 to 2023. Various Mg-based materials have been evaluated in animal research, and the application of pure Mg and Mg alloys have been reported in clinical follow-ups involving multiple anatomical sites and musculoskeletal disorders. Soft tissue and intraosseous PIGA are common in both animal tests and clinical follow-ups, and potentially associated with certain adverse events. Radiological examinations especially micro-CT and clinical CT scans provide valuable information for quantitative and longitudinal analysis. While according to simulation tests involving Mg implantation and chemical processing, tissue fixation could lead to an increase in the volume of gas cavity, thus the results obtained from ex vivo imaging or histopathological evaluations should be interpreted with caution. There still lacks standardized procedures or consensus for both preclinical and clinical evaluation of PIGA. However, by providing focused insights into the topic, this evidence-based study will facilitate future animal tests and clinical evaluations, and support developing biocompatible Mg implants for the treatment of musculoskeletal disorders

Surgical Classification for Preclinical Rat Femoral Bone Defect Model: Standardization Based on Systematic Review, Anatomical Analysis and Virtual Surgery

Though surgical techniques profoundly influence in vivo experiments, significant heterogeneity exists in current surgeries for inducing rat femoral bone defects. Such variations reduce the reproducibility and comparability of preclinical studies, and are detrimental to clinical translation. The purposes of this study were: (1) to conduct a systematic review of rat femoral defect models, summarizing and analyzing the surgical techniques; (2) to analyze surgical design and potential pitfalls via 3D anatomy and virtual surgeries for fostering future precision research; and (3) to establish a surgical classification system, for improving the reproducibility and comparability among studies, avoiding unnecessary repetitive experiments. The online database PubMed was searched to identify studies from January 2000 to June 2022 using keywords, including rat, femur, bone defect. Eligible publications were included for a review of surgical methods. Anatomical analysis and virtual surgeries were conducted based on micro-CT reconstruction of the rat femur for further investigation and establishment of a classification system. A total of 545 publications were included, revealing marked heterogeneity in surgical methods. Four major surgical designs were reported for inducing defects from the proximal to distal femur: bone tunnel, cortical window, segmental defect, and wedge-shaped defect. Anatomical analysis revealed potential pitfalls hindering efficient clinical translation. A classification system was established according to the anatomical region, surgical design, and fixation devices. This systematic review in combination with 3D analysis and virtual surgery provides a general overview of current surgical approaches to inducing femoral defects in rats, and establishes a surgical classification facilitating preclinical research of quality and translational value

Systemic modulation of skeletal mineralization by magnesium implant promoting fracture healing: Radiological exploration enhanced with PCA-based machine learning in a rat femoral model

The clinical application of magnesium (Mg) and its alloys for bone fractures has been well supported by in vitro and in vivo trials. However, there were studies indicating negative effects of high dose Mg intake and sustained local release of Mg ions on bone metabolism or repair, which should not be ignored when developing Mg-based implants. Thus, it remains necessary to assess the biological effects of Mg implants in animal models relevant to clinical treatment modalities. The primary purpose of this study was to validate the beneficial effects of intramedullary Mg implants on the healing outcome of femoral fractures in a modified rat model. In addition, the mineralization parameters at multiple anatomical sites were evaluated, to investigate their association with healing outcome and potential clinical applications. Compared to the control group without Mg implantation, postoperative imaging at week 12 demonstrated better healing outcomes in the Mg group, with more stable unions in 3D analysis and high-mineralized bridging in 2D evaluation. The bone tissue mineral density (TMD) was higher in the Mg group at the non-operated femur and lumbar vertebra, while no differences between groups were identified regarding the bone tissue volume (TV), TMD and bone mineral content (BMC) in humerus. In the surgical femur, the Mg group presented higher TMD, but lower TV and BMC in the distal metaphyseal region, as well as reduced BMC at the osteotomy site. Principal component analysis (PCA)-based machine learning revealed that by selecting clinically relevant parameters, radiological markers could be constructed for differentiation of healing outcomes, with better performance than 2D scoring. The study provides insights and preclinical evidence for the rational investigation of bioactive materials, the identification of potential adverse effects, and the promotion of diagnostic capabilities for fracture healing

Multicolor Histochemical Staining for Identification of Mineralized and Non-Mineralized Musculoskeletal Tissue: Immunohistochemical and Radiological Validation in Decalcified Bone Samples

Histochemical staining of paraffin-embedded decalcified bone samples is commonly used in preclinical research of musculoskeletal diseases, enabling the visualization of multiple tissue components by the application of chromogens. The purpose of this study was to introduce a novel multicolor staining protocol involving optimized chemical reagents and procedure, allowing the identification of high-mineralized bone, low-mineralized fracture callus, cartilage and skeletal muscle fibers simultaneously. Fractured femur and healthy tail vertebra samples from adult male Sprague–Dawley rats were decalcified with EDTA and formic acid, respectively, followed by paraffin embedding, tissue sectioning and multicolor staining. Conventional Movat’s pentachrome and safranin O / fast green staining were conducted in parallel for comparison. Immunohistochemical staining of collagen type-X and micro-CT analysis were included to further validate the efficacy of the staining method. The multicolor staining allowed visualization of major musculoskeletal tissue components in both types of decalcified samples, providing quality outcomes with fewer chemical reagents and simplified procedures. Immunohistochemical staining demonstrated its capacity for identification of the endochondral ossification process during fracture healing. Micro-CT imaging validated the staining outcome for high-mineralized skeletal tissue. The application of the multicolor staining may facilitate future preclinical research involving decalcified paraffin-embedded samples