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

Suture Expansion Mice에서 인장 하중을 받는 뼈 리모델링의 3D 시각화

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.

저자 스포트라이트: 뼈 리모델링을 시각화하기 위한 PEGASOS 조직 투명화 기법 

봉합사 확장 마우스 모델의 뼈 리모델링을 위한 3D 시각화 기법

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  

마우스 봉합사 확장 모델을 준비하기 위한 시상 봉합사 확장 수술

먼저 두 개의 리텐션 홀더를 준비합니다. 가벼운 와이어 플라이어와 0.014인치 호주 와이어를 사용하여 나선형 루프를 만듭니다. 그런 다음 맞춤형 스테인리스 스틸 스프링을 준비하고 스프링을 적절한 길이로 자릅니다.또한 길이가 7mm인 직선 호주 철사 또는 직선 강선 1개를 준비하고 장벽으로 사용할 직경 2mm의 종이 두 장을 자릅니다. 그런 다음 마취된 쥐를 수술대에 놓고 제모 크림이나 면도기로 머리 위의 털을 조심스럽게 제거합니다. 그런 다음 요오드포름과 75% 알코올을 번갈아 가며 사용하여 쥐의 두피를 소독합니다.마우스의 머리가 위쪽에 오도록 놓고 수술용 테이프를 사용하여 팔다리를 수술대에 고정합니다. 이제 수술용 가위를 사용하여 쥐의 목 근처 두피를 따라 아치형 플랩을 수행하여 시상 봉합사와 주변 두개골을 완전히 노출시킵니다. 그런 다음 수술대에 6-0 봉합사로 두피 플랩을 고정합니다.두개골을 말리고 37% 인산으로 20초 동안 에칭합니다. 그런 다음 생리 식염수를 사용하여 산성 에칭을 청소하십시오. 두 개의 홀더를 시상 봉합사에서 3mm 떨어진 정수리 뼈의 양쪽에 놓고 광 경화 접착제로 붙입니다.그런 다음 두피 플랩을 다시 끼웁니다. 홀더의 위치에 라벨을 붙이고 해당 위치에서 두피에 두 개의 작은 구멍을 뚫습니다. 피부 표면을 노출시키기 위해 구멍을 통해 작은 고리를 동시에 통과시켜 플랩 두피를 재설정합니다.스프링과 가이드 와이어를 설치하려면 스프링을 두 홀더 사이의 거리보다 1mm 더 길게 자릅니다. 압력 스프링을 압축하여 양쪽의 작은 코일 사이에 놓습니다. 스테인리스강을 작은 코일과 스프링에 통과시키고 스프링을 풀어 약 30G의 시작 추력을 얻습니다.그런 다음 스프링과 홀더 사이에 두 개의 종이 조각을 놓고 광경화 접착제로 접착하여 스프링의 양쪽 끝에 장벽을 설정합니다. 확장 후 마이크로 CT를 사용하여 두개골 봉합사의 변화를 시각화했습니다. 대조군과 비교했을 때, 두개골 봉합사는 하루 동안 힘을 가한 후 점차적으로 크게 확장되었고, 5일째 이후에는 뼈 가장자리에 솜털 같은 뼈 돌출부가 나타났다.또한, 이중 표지 방법을 이용한 광물화의 시각화는 하중 부하가 골형성을 유의하게 활성화시키는 것을 보여주었습니다. 7일 후 새로 광물화된 뼈가 관찰되었는데, 이는 봉합사 확장 후 뼈 리모델링 과정을 나타냅니다.

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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 ...