Name: PEGASOS Method to Prepare Transparent Tissues and Calvarial Bones
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

3D Visualization of Bone Remodeling Under Tensile Load in Suture Expansion Mice

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.

Author Spotlight: PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling 

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

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

PEGASOS Method to Prepare Transparent Tissues and Calvarial Bones

To begin, fix the limbs of an anesthetized mouse with adhesive tape. Hold the skin of the abdomen upward and carefully cut along the circumference of the thorax to fully expose it. Make a small cut in the right atrium with ophthalmic scissors and immediately insert a perfusion 22 gauge needle at the apex of the left ventricle.Then push 30 to 50 milliliters of cardiac perfusion fluid through the syringe. After the outflow fluid from the right atrium becomes completely clear, infuse an equal volume of 4%PFA solution. Dissect the tissues and organs and fix them overnight with 4%PFA at four degrees Celsius.To induce tissue decalcification. place the fixed heart tissue in 10 milliliters of EDTA solution on a shaking table at 37 degrees Celsius. Incubate for about two days and change the fluid daily.Place the fixed calvaria bones in 20 milliliters of 25%Quadrol solution for tissue decolorization. Incubate on a shaking table at 37 degrees Celsius for one day and change the fluid once. Next, place the tissue sequentially in 30%Tertiary-Butanol or TB solution.Then 50%TB solution, and then 70%TB solution to perform gradient degreasing. Subsequently dehydrate the samples and TB PEG solution for six hours on a shaking table at 37 degrees Celsius. Finally, place the completely dehydrated tissue in BB PEG solution on a shaking table at 37 degrees Celsius.After two to four hours of incubation, the tissue will turn transparent. The PEGASOS Tissue Clearing Method resulted in transparent tissue. Cleared tissue with whole mount EDU staining showed that the labeling was efficient and well preserved.In the control group, several EDU positive cells were diffusely distributed throughout the sutures. After one day of suture expansion, proliferating cells were observed in the middle and edges of the suture. As the suture widened, the number of proliferating cells decreased over time.The green labeled cells were small round cells in the bone marrow, which differed from the EDU positive suture cells.

Research

JoVE Journal

Biology

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