Name: Méthode PEGASOS pour préparer les tissus transparents et les os calvaires
Uploaded: 2023-08-18T12:00:00
Description: Watch this Scientific Journal Video about PEGASOS Method to Prepare Transparent Tissues and Calvarial Bones at JoVE.com

pegasos method to prepare transparent tissues and calvarial bones

Visualisation 3D du remodelage osseux sous charge de traction chez des souris à expansion de suture

Craniofacial sutures are fibrous tissues that connect craniofacial bones and play essential roles in the growth and remodeling of craniofacial bones. The structure of the suture resembles a river, providing a flow of cell resources to nourish and build the &#34;river bank&#34;, known as the osteogenic fronts, which contribute to the formation of craniofacial bones via intramembranous osteogenesis1.
Interest in craniofacial sutures has been driven by clinical needs to understand premature closure of cranial sutures and facial suture dysfunction, which may lead to craniofacial deformities and even life-threatening conditions in children. Open suturectomy is routinely used in clinical treatment, but long-term follow-up has shown incomplete re-ossification recurrence in some patients2. Minimally invasive craniotomy assisted by expansion springs or endoscopic stripe craniectomy may provide a safer approach to preserving the potential suture rather than discarding the tissues3. Similarly, orthopedic therapies such as facemasks and expansion appliances have been widely used to treat sagittal or horizontal maxillary hypoplasia, with some studies extending the age limitation to treat adult patients via miniscrew-assisted palatal expanders4,5,6. Additionally, cranial suture regeneration with mesenchymal stem cells (MSCs) combined with biodegradable materials is a potential therapy in the future, offering a novel direction for the treatment of related diseases7. However, the function process or regulatory mechanism of sutures remains elusive.
Bone remodeling mainly consists of a balance between bone formation conducted by osteoblasts and bone resorption conducted by osteoclasts, where osteogenic differentiation of stem cells stimulated by mechanical signals plays an important role. After decades of research, it has been found that craniofacial sutures are highly plastic mesenchymal stem cell niches8. Suture stem cells (SuSCs) are a heterogeneous group of stem cells, belonging to mesenchymal stem cells (MSCs) or bone stem cells (SSCs). SuSCs are labeled in vivo by four markers, including Gli1, Axin2, Prrx1, and Ctsk. Gli1+ SuSCs, in particular, have strictly verified the biological characteristics of stem cells, not only exhibiting high expression of typical MSC markers but also demonstrating excellent osteogenic and chondrogenic potential9. Previous research has shown that Gli1+ SuSCs actively contribute to new bone formation under tensile force, identifying them as the suture stem cell source supporting distraction osteogenesis10.
In the past, extensive mechanical characteristics of stem cells were studied in vitro via Flexcell, four-point bending, micro-magnet loading system, and others. Although mouse cranial suture-derived mesenchymal cells have been identified in vitro11, and human suture mesenchymal stem cells have also been isolated recently12, the biomechanical response of suture cells remains unclear in the in vitro system. To further investigate the bone remodeling process, a suture expansion model based on isolated calvaria organ culture has been established, paving the way for establishing a useful in vivo suture expansion model1,13. Rabbits14 and rats15 have been the most widely used animals in basic research for suture expansion. However, mice are preferred animal models for exploring human disease due to their highly homologous genome with humans, numerous gene modification lines, and strong reproductive hybridization ability. Existing mouse models of cranial suture expansion typically rely on stainless steel orthodontic spring wires to apply tensile force to the sagittal suture16,17. In these models, two holes are made in each side of the parietal bones to fix the expansion device, and the wires are embedded under the skin, which may affect the cell activation mode.
Regarding the visualization method, the two-dimensional observation of slices in the sagittal direction has been generally adopted for decades. However, considering that bone remodeling is a complex three-dimensional dynamic process, obtaining complete three-dimensional information has become an urgent need. The PEGASOS tissue transparency technique emerged to meet this requirement18,19. It offers unique advantages for the transparency of hard and soft tissues, enabling the complete bone remodeling process to be reproduced in three-dimensional space.
To gain a deeper and more comprehensive understanding of the physiological changes in the bone remodeling periods, a standard sagittal suture expansion mouse model with a spring setting between the handmade holders was established10. With a standardized acid etching and bonding procedure, the expansion device could be firmly bonded to the cranial bone, generating a tensile force perpendicular to the sagittal suture. Furthermore, the PEGASOS tissue clearing method was applied after double labeling of the mineralized bone post-expansion to fully visualize the bone modeling changes after suture expansion.

Pleins feux sur l’auteur : Technique de nettoyage des tissus PEGASOS pour visualiser le remodelage osseux 

Technique de visualisation 3D pour le remodelage osseux dans un modèle murin d’expansion de suture

The interest in craniofacial sutures has been driven by the clinical needs to understand the mechanisms of orthodontic and the orthopedic treatment. The scope of our study is to establish a standardized suture expansion mouse model, and the 3D visualization method to study the mechanobiological changes of suture and bone remodeling upon expansion. PEGASOS tissue clearing technique has been combined with various labeling methods to obtain versatile information of suture and bond remodeling in space perspective.This model is suitable for various strengths of transgenic mice of different age. It is convenient to adjust the magnitude of tensile stress and withdraw force at any time. With the PEGASOS technique, we can observe the three dimensional spatial temporal distribution of cranial suture stem cells during bone remodeling.With the PEGASOS technique, we will further explore the relationship between suture stem cells and mechanical stress, as well as its specific mechanisms.

Watch this Scientific Journal Video about PEGASOS Method to Prepare Transparent Tissues and Calvarial Bones at JoVE.com

Méthode PEGASOS pour préparer les tissus transparents et les os calvaires 

Pour commencer, fixez les membres d’une souris anesthésiée avec du ruban adhésif. Tenez la peau de l’abdomen vers le haut et coupez soigneusement le long de la circonférence du thorax pour l’exposer complètement. Faites une petite incision dans l’oreillette droite avec des ciseaux ophtalmiques et insérez immédiatement une aiguille de perfusion de calibre 22 à l’apex du ventricule gauche.Poussez ensuite 30 à 50 millilitres de liquide de perfusion cardiaque à travers la seringue. Une fois que le liquide d’écoulement de l’oreillette droite devient complètement clair, perfusez un volume égal de solution à 4 % de PFA. Disséquez les tissus et les organes et fixez-les pendant la nuit avec 4 % de PFA à quatre degrés Celsius.Pour induire la décalcification des tissus. placer le tissu cardiaque fixe dans 10 millilitres de solution d’EDTA sur une table agitée à 37 degrés Celsius. Incubez pendant environ deux jours et changez le liquide tous les jours.Placez les os calvaires fixes dans 20 millilitres de solution 25%Quadrol pour la décoloration des tissus. Incuber sur une table vibrante à 37 degrés Celsius pendant une journée et changer le liquide une fois. Ensuite, placez le tissu séquentiellement dans une solution de butanol tertiaire ou de tuberculose à 30 %.Ensuite, une solution de 50 % de To, puis une solution de 70 % de To pour effectuer un dégraissage de gradient. Ensuite, déshydrater les échantillons et la solution de TB PEG pendant six heures sur une table agitée à 37 degrés Celsius. Enfin, placez le tissu complètement déshydraté dans une solution de BB PEG sur une table agitée à 37 degrés Celsius.Après deux à quatre heures d’incubation, le tissu deviendra transparent. La méthode d’élimination des tissus PEGASOS a permis d’obtenir des tissus transparents. Les tissus nettoyés avec coloration EDU à montage entier ont montré que le marquage était efficace et bien conservé.Dans le groupe témoin, plusieurs cellules positives de l’EDU étaient distribuées de manière diffuse dans les sutures. Après une journée d’expansion de la suture, des cellules proliférantes ont été observées au milieu et sur les bords de la suture. Au fur et à mesure que la suture s’élargissait, le nombre de cellules proliférantes diminuait avec le temps.Les cellules marquées en vert étaient de petites cellules rondes dans la moelle osseuse, qui différaient des cellules de suture positives de l’EDU.

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Biology

PEGASOS Method to Prepare Transparent Tissues and Calvarial Bones

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...