Name: 透明組織と頭蓋骨を調製するPEGASOS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

縫合糸拡張マウスにおける引張荷重下における骨リモデリングの3D可視化

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.

著者スポットライト:骨リモデリングを視覚化するためのPEGASOS組織透明化技術 

縫合糸拡張マウスモデルにおける骨リモデリングのための3次元可視化技術

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

透明組織と頭蓋骨を調製するPEGASOS法 

まず、麻酔をかけたマウスの手足を粘着テープで固定します。腹部の皮膚を上に保持し、胸部の円周に沿って慎重に切り取り、完全に露出させます。眼科用ハサミで右心房に小さな切り込みを入れ、すぐに左心室の頂点に灌流22ゲージの針を挿入します。次に、30〜50ミリリットルの心灌流液をシリンジに押し込みます。.右心房からの流出液が完全に透明になったら、等量の4%PFA溶液を注入します。組織や臓器を解剖し、摂氏4度で4%PFAで一晩固定します。組織の脱灰を誘導する。固定された心臓組織を10ミリリットルのEDTA溶液に入れ、摂氏37度の振とう台に置きます。約2日間インキュベートし、毎日液体を交換します。固定された頭蓋骨を20ミリリットルの25%Quadrol溶液に入れて、組織の脱色を行います。摂氏37度の振動台で1日インキュベートし、液体を1回交換します。次に、組織を30%三級ブタノールまたは結核溶液に順次入れます。次に 50%TB 溶液、次に 70%TB 溶液でグラジエント脱脂を行います。続いて、サンプルとTB PEG溶液を摂氏37度の振とう台で6時間脱水します。最後に、完全に脱水した組織をBB PEG溶液に入れ、摂氏37度の振とう台に置きます。2〜4時間のインキュベーション後、組織は透明になります。PEGASOS Tissue Clearing Methodにより、透明な組織が得られました。ホールマウントEDU染色で透明化した組織は、標識が効率的で保存状態が良好であることを示しました。対照群では、いくつかのEDU陽性細胞が縫合糸全体にびまん性に分布していた。縫合糸の拡張の1日後、縫合糸の中央と端に増殖細胞が観察されました。縫合糸が広がるにつれて、増殖する細胞の数は時間の経過とともに減少しました。緑色で標識された細胞は、骨髄の小さな丸い細胞であり、EDU陽性の縫合糸細胞とは異なりました。

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 ...