Name: Metodo PEGASOS per la preparazione di tessuti trasparenti e ossa calvari
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

Visualizzazione 3D del rimodellamento osseo sotto carico di trazione in topi ad espansione di sutura

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.

Autore in primo piano: Tecnica di pulizia dei tessuti PEGASOS per visualizzare il rimodellamento osseo 

Una tecnica di visualizzazione 3D per il rimodellamento osseo in un modello murino di espansione della sutura

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

Metodo PEGASOS per la preparazione di tessuti trasparenti e ossa calvari 

Per iniziare, fissa gli arti di un topo anestetizzato con del nastro adesivo. Tenere la pelle dell'addome verso l'alto e tagliare con cura lungo la circonferenza del torace per esporla completamente. Praticare un piccolo taglio nell'atrio destro con le forbici oftalmiche e inserire immediatamente un ago di perfusione calibro 22 all'apice del ventricolo sinistro.Quindi spingere da 30 a 50 millilitri di liquido di perfusione cardiaca attraverso la siringa. Dopo che il fluido in uscita dall'atrio destro diventa completamente limpido, infondere un volume uguale di soluzione di PFA al 4%. Sezionare i tessuti e gli organi e fissarli durante la notte con PFA al 4% a quattro gradi Celsius.Per indurre la decalcificazione dei tessuti. posizionare il tessuto cardiaco fisso in 10 millilitri di soluzione EDTA su un tavolo vibrante a 37 gradi Celsius. Incubare per circa due giorni e cambiare il liquido ogni giorno.Mettere le ossa della calvaria fissate in 20 millilitri di soluzione di Quadrol al 25% per la decolorazione dei tessuti. Incubare su un tavolo vibrante a 37 gradi Celsius per un giorno e cambiare il fluido una volta. Successivamente, posizionare il tessuto in sequenza in una soluzione di butanolo terziario al 30% o TB.Quindi soluzione al 50% di TB e quindi soluzione al 70% di TB per eseguire lo sgrassaggio a gradiente. Successivamente, disidratare i campioni e la soluzione di PEG TB per sei ore su un tavolo vibrante a 37 gradi Celsius. Infine, posizionare il tessuto completamente disidratato in una soluzione di BB PEG su un tavolo vibrante a 37 gradi Celsius.Dopo due o quattro ore di incubazione, il tessuto diventerà trasparente. Il metodo PEGASOS Tissue Clearing ha portato a un tessuto trasparente. Il tessuto eliminato con colorazione EDU a monte intero ha dimostrato che l'etichettatura era efficiente e ben conservata.Nel gruppo di controllo, diverse cellule positive all'EDU sono state distribuite in modo diffuso in tutte le suture. Dopo un giorno di espansione della sutura, sono state osservate cellule proliferanti al centro e ai bordi della sutura. Man mano che la sutura si allargava, il numero di cellule proliferanti diminuiva nel tempo.Le cellule marcate in verde erano piccole cellule rotonde nel midollo osseo, che differivano dalle cellule di sutura positive all'EDU.

Research

JoVE Journal

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 ...