sagittal suture expansion surgery to prepare mouse suture expansion model

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 Sagittal Suture Expansion Surgery to Prepare Mouse Suture Expansion Model at JoVE.com

Sagittal Suture Expansion Surgery to Prepare Mouse Suture Expansion Model  

Begin by preparing two retention holders. Use light wire pliers and a 0.014 inch Australian wire to make a helical loop. Then prepare customized stainless steel springs and cut the spring into an appropriate length.Also prepare one straight Australian wire or straight steel wire of length seven millimeters and cut two pieces of paper with diameters of two millimeters to use as barriers. Next, place an anesthetized mouse on the operating table and carefully remove the fur on top of the head with hair removal cream or a shaver. Then disinfect the scalp of the mouse using alternating rounds of Iodoform and 75%alcohol.Position the mouse with its head on the top and use surgical tapes to fix the limbs on the operating table. Now, use surgical scissors to perform an arcuate flap along the scalp near the neck of the mouse, fully exposing the sagittal suture and surrounding skull. Then, fix the scalp flap with a 6-0 suture on the operating table.Dry the skull and etch it with 37%phosphoric acid for 20 seconds. Then use normal saline to clean up the acid etching. Place the two holders on both sides of the parietal bones, three millimeters from the sagittal sutures and glue them with a light cured adhesive.Then put back the scalp flap. Label the position of the holders and cut two small holes in the scalp at the corresponding positions. Reset the flap scalp by simultaneously passing the small loops through the holes to expose the skin surface.To install the spring and guide wire, cut the spring one millimeter longer than the distance between the two holders. Compress the pressure spring and place it between the small coils on both sides. Pass the stainless steel wire through the small coils and the spring and release the spring to obtain a starting thrust of about 30G.Next, place two scraps of paper between the spring and the holders and bond them with a light cured adhesive to set up barriers at both ends of the spring. After expansion, micro CT was used to visualize the changes in the cranial suture. In comparison to controls, the cranial suture gradually and significantly expanded after applying force for one day and fluffy bony protrusions appeared on the bone edge after day five.Also, visualization of mineralization using double labeling method, showed that force loading significantly activated osteogenesis. Newly mineralized bone was observed after seven days, indicating the bone remodeling process after suture expansion.

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Research

JoVE Journal

Biology

150 Görüntüleme.  Shanghai Jiao Tong University School of Medicine. İki saklama tutucu hazırlayarak başlayın. Sarmal bir döngü yapmak için hafif tel pense ve 0.014 inç Avustralya teli kullanın. Ardından özelleştirilmiş paslanmaz çelik yaylar hazırlayın ve yayı uygun uzunlukta kesin.Ayrıca yedi milimetre uzunluğunda bir düz Avustralya teli veya düz çelik tel hazırlayın ve bariyer olarak kullanmak için iki milimetre çapında iki kağıt parçası kesin. Daha sonra, ameliyat masasına anestezi uygulanmış bir fare yerleştirin v...