Name: Sagittal Suture Expansion Surgery to Prepare Mouse Suture Expansion Model
Uploaded: 2023-08-18T12:00:00
Description: 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

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

Sagittal Suture Expansion Surgery to Prepare Mouse Suture Expansion Model  

矢状缝合扩张手术准备小鼠缝合扩张模型

首先准备两个固定支架。使用轻线钳和 0.014 英寸的澳大利亚线制作螺旋环。然后准备定制的不锈钢弹簧，并将弹簧切割成合适的长度。还要准备一根长 7 毫米的澳大利亚直线或直钢丝，并剪下两张直径为 2 毫米的纸作为屏障。接下来，将麻醉的小鼠放在手术台上，用脱毛膏或剃须刀小心地去除头顶的毛发。然后使用交替的碘仿和75%酒精对小鼠的头皮进行消毒。将鼠标的头放在顶部，并使用手术胶带将四肢固定在手术台上。现在，使用手术剪刀沿着小鼠颈部附近的头皮进行弓形皮瓣，完全暴露矢状缝线和周围的颅骨。然后，在手术台上用 6-0 缝合线固定头皮瓣。干燥头骨并用 37% 磷酸蚀刻 20 秒。然后用生理盐水清理酸蚀。将两个支架放在顶骨的两侧，距离矢状缝合线三毫米，并用轻固化的粘合剂粘合它们。然后放回头皮瓣。标记支架的位置，并在相应位置的头皮上切两个小孔。通过同时将小环穿过孔以露出皮肤表面来重置皮瓣头皮。要安装弹簧和导丝，请将弹簧切割得比两个支架之间的距离长一毫米。压缩压力弹簧并将其放置在两侧的小线圈之间。将不锈钢丝穿过小线圈和弹簧，松开弹簧，以获得约30G的起动推力。接下来，在弹簧和支架之间放置两张纸片，并用轻固化的粘合剂将它们粘合起来，以在弹簧的两端设置屏障。扩张后，显微CT可视化颅缝线的变化。与对照组相比，用力1天后颅缝逐渐明显扩大，第5天后骨缘出现蓬松的骨突起。此外，使用双重标记方法对矿化进行可视化，表明力载荷显着激活了成骨作用。7 天后观察到新矿化的骨，表明缝合线扩张后的骨重塑过程。

Research

JoVE Journal

Biology

Sagittal Suture Expansion Surgery to Prepare Mouse Suture Expansion Model

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 ...