Name: PEGASOS制备透明组织和颅骨的方法
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

缝合扩张小鼠拉伸载荷下骨重塑的 3D 可视化

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.

作者聚焦：PEGASOS 组织透明化技术可视化骨骼重塑 

缝合扩张小鼠模型中骨重塑的 3D 可视化技术

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制备透明组织和颅骨的方法 

首先，用胶带固定麻醉小鼠的四肢。握住腹部皮肤向上，沿着胸部的圆周小心地切开，使其完全暴露。用眼科剪刀在右心房切一个小口，并立即在左心室的顶点插入灌注 22 号针头。然后将 30 至 50 毫升心脏灌注液推入注射器。从右心房流出的液体完全透明后，输注等体积的 4%PFA 溶液。解剖组织和器官，并在4摄氏度下用4%PFA固定过夜。诱导组织脱钙。将固定的心脏组织放入 10 毫升 EDTA 溶液中，放在 37 摄氏度的摇床上。孵育约两天，每天更换液体。将固定的颅骨放入 20 毫升 25%Quadrol 溶液中进行组织脱色。在37摄氏度的摇床上孵育一天，并更换一次液体。接下来，将组织依次放入 30% 叔丁醇或 TB 溶液中。然后是50%TB溶液，然后是70%TB溶液进行梯度脱脂。随后将样品和TB PEG溶液在37摄氏度的摇床上脱水6小时。最后，将完全脱水的组织置于BB PEG溶液中，放在37摄氏度的摇床上。孵育两到四个小时后，组织将变成透明。PEGASOS组织清除法得到透明的组织。使用全贴片 EDU 染色的透明组织表明标记有效且保存完好。在对照组中，几个EDU阳性细胞弥漫分布在整个缝合线中。缝合线扩张一天后，在缝合线的中间和边缘观察到增殖细胞。随着缝合线的扩大，增殖细胞的数量随着时间的推移而减少。绿色标记的细胞是骨髓中的小圆形细胞，与EDU阳性缝合细胞不同。

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 ...