creating calvarial suture-bony composite defects in rats and postoperative healing dynamics

Standardizing Rat Calvarial Suture-Bony Defect Model for Regenerative Studies

Cranial sutures are dense fibrous connections between cranial bones, acting as joints to facilitate slight skull movement and providing a protective cushion for the brain under pressure1. In recent years, increased research has highlighted the pivotal role of cranial sutures in skull development, craniofacial homeostasis, and inherent osteo-reparative potential2,3,4,5,6,7,8. During periods of growth and development, cranial sutures act as the main growth centers in the skull4. New bone formation occurs at the osteogenic fronts on both sides of the sutures, while the cells within the sutures maintain an undifferentiated mesenchymal state, ensuring balanced skull expansion alongside brain growth1. The loss of cranial sutures at this time results in a discrepancy between the growth of the skull and brain, leading to severe issues like brain injuries, hydrocephalus, increased intracranial pressure, and cognitive dysfunction3,9.
Besides, cranial sutures play a crucial role in determining the prognosis of calvarial defects5,7,10. The regenerative potential across the calvarial surface is unevenly distributed, with cranial sutures showing remarkably superior capabilities compared to non-suture regions10,11. One study indicates that the speed of calvarial defect healing inversely correlates with the distance between the cranial suture and the injury site10. Specifically, the removal of coronal and sagittal sutures leads to the non-healing of parietal bone defects7, emphasizing the necessity of suture regeneration in calvarial defects. However, the focus of the current studies is predominantly on the restoration of cranial osseous structure while neglecting the regeneration of suture mesenchyme.
Regarding advancements in suture regeneration, promising outcomes have been observed with the transplantation of suture-containing bone flaps, mesenchymal stem cells (MSCs), and artificial biomaterials. When bone flaps with sutures were transplanted into calvarial defects, they successfully integrated and healed, in contrast to those without sutures displaying non-union and an inability to form periosteum, dura mater, or osteocytes5. Likewise, the implantation of MSCs derived from bone marrow into sagittal suture-bony composite defects facilitated the formation of suture-like gaps12. Of note, a recent study highlighted the realization of suture regeneration with Gli1+ MSCs, allowing for intracranial pressure control, skull deformity correction, and enhanced neurocognitive function13. As regenerative medicine and biomedical engineering develop, researchers increasingly focus on tissue engineering biomaterials due to their adaptable and customizable characteristics14. Notably, polytetrafluoroethylene membranes have been proven to be effective in reconstructing cranial bone and suture mesenchyme simultaneously15,16.
However, craniofacial research lacks established models for exploring suture mesenchymal regenerative therapies, unlike relatively mature models in repairing other tissues such as bone, skin, cartilage, and muscles17. The absence of a standardized model constrains the study of suture-regenerative therapies and makes it challenging to perform comparative analysis across different studies. Therefore, our study established a practicable and reproducible rat calvarial suture-bony composite defect. Through this method, we aim to develop appropriate clinical interventions for cranial suture reconstruction, offering novel perspectives on the functional repairment of calvarial defects and decreasing unfavorable outcomes resulting from suture loss.

Author Spotlight: Development and Evaluation of a Standardized Rat Model for Calvarial Suture-Bony Composite Defects

The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation

Our research focuses on the detailed surgical procedures of calvarial suture-bony composite defects in rats alongside the investigations into the short-term and long-term prognoses of the model. The primary objective is to establish a standardized model that can be utilized for advancing suture regenerative therapies. For the current animal model, the primary challenge lies in completely removing the coronal suture while preserving the sagittal and frontal sutures with minimal osseous tissue removal.Our protocol allows for more straightforward comparation through self-control by creating defects in both the left and the right halves of the coronal suture and applying different interventions to each side.

Creating Calvarial Suture-Bony Composite Defects in Rats and Postoperative Healing Dynamics

To begin, place the anesthetized rat in a prone position with the head naturally extending vertically. Using an electric shaver, remove the hair from the scalp, between the nasal bridge and the cervical spine joint. Disinfect the surgical area in circular motions, radiating from the center with 2%iodophor, followed by 75%ethanol.Employ a surgical drape to cover the dorsum of the animal and the fur surrounding the incision. Starting from the midpoint of the nasal bone, with a disposable scalpel, make a two centimeter longitudinal skin incision following the midline of the cranium. Using a scalpel, make a midline periosteal incision, mirroring the initial point and extent of the skin layer.Then gently lift the periosteum on both sides of the incision with a periosteal elevator to expose parietal bones, frontal bones, and coronal sutures. Apply vertical force with a 1.2 millimeter diameter round burr on the coronal suture until a sense of breakthrough is felt. From the penetration point, move the bur laterally along the coronal suture to create an approximately four millimeter long positioning groove.Then remove bone tissue with the bur on both sides of the positioning groove to initially form a rectangular full thickness defect. Employ a 0.8 millimeter diameter round burr to refine details involving the grinding of right angles and the smoothing of defect margins. Create two defects across the left and right halves of the coronal suture for self comparison.Regularly check the length and width of the defects using a Vernier caliper to ensure consistency across all samples. After surgery, close the skin with three zero monofilament sutures. Micro CT images, and cross-sectional views of suture bony composite defects at postoperative day zero Confirmed the successful creation of a full thickness calvarial defect.Micro CT images, and the corresponding statistical analysis at six weeks post-operation revealed a natural inclination towards defect closure with more pronounced tendencies 12 weeks after surgery, indicating potential suture fusion over time. Histological analysis using hematoxylin and eosin and Masson's trichrome staining two weeks postoperatively showed extension and continuity of the periosteum and dura mater, forming dense fibrous tissue that sealed the defect. By 12 weeks, large pieces of new bone were observed in the defect center.

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Research

JoVE Journal

Medicine

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development (the latter occurs in the developing cranium). However, the lack of a standardized model hinders progress in investigating suture-regenerative therapies and poses challenges for conducting comparative analyses across distinct studies. To address this issue, the current protocol describes the detailed modeling process of calvarial suture-bony composite defects in rats.
The model was generated by drilling full-thickness rectangular holes measuring 4.5 mm &#215; 2 mm across the coronal sutures. The rats were euthanized, and the cranium samples were harvested postoperatively at day 0, week 2, week 6, and week 12. &#181;CT results from samples collected immediately post-surgery confirmed the successful establishment of the suture-bony composite defect, involving the removal of the coronal suture and the adjacent bone tissues.
Data from the 6th and 12th postoperative weeks demonstrated a natural healing tendency for the defect to close. Histological staining further validated this trend by showing increased mineralized fibers and new bone at the defect center. These findings indicate progressive suture fusion over time following calvarial defects, underscoring the significance of therapeutic interventions for suture regeneration. We anticipate that this protocol will facilitate the development of suture-regenerative therapies, offering fresh insights into the functional restoration of calvarial defects and reducing adverse outcomes associated with suture loss.

We describe the detailed surgical procedures of calvarial suture-bony composite defects in rats, alongside the investigations into the short-term and long-term prognoses of the model. We aim to construct a standardized model for developing suture-regenerative therapies.

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development ...