This study was supported by the National Natural Science Foundation of China 82100982 (F.L.), 82101000 (H.W.), 82001019 (B.Y.), Science and Technology Department of Sichuan Province 2022NSFSC0598 (B.Y.), 2023NSFSC1499 (H.W.) and Research Funding from West China School/Hospital of Stomatology Sichuan University (RCDWJS2021-5). Figure 3 was created with Biorender.com.

All animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2021-597). A total of 12 (3 rats at each of the four time points) Sprague-Dawley (SD) rats (male, 300 g, 8 weeks old) were obtained from a commercial source (see Table of Materials).
1. Presurgical preparation
<ol>
	<li>Surgical items
	<ol>
		<li>Prepare surgical instruments displayed in <strong class=...

Standardizing Rat Calvarial Suture-Bony Defect Model for Regenerative Studies

<ol>
	<li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+suture+mesenchymal+stem+cells:+insights+and+advances.">Cranial suture mesenchymal stem cells: insights and advances.</a> Biomolecules. 11 (8), 1129 (2021).</li><li>Opperman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+sutures+as+intramembranous+bone+growth+sites.">Cranial sutures as intramembranous bone growth sites.</a> Dev Dyn. 219 (4), 472-485 (2000).</li><li>Lenton, K. A., Nacamuli, R. P., Wan, D. C., Helms, J. A., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+suture+biology.">Cranial suture biology.</a> Curr Top Dev Biol. 66 (4), 287-328 (2005).</li><li>Slater, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+sutures:+A+brief+review.">Cranial sutures: A brief review.</a> Plast Reconstr Surg. 121 (4), 170-178 (2008).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+suture+provides+a+niche+for+mesenchymal+stem+cells+of+craniofacial+bones.">The suture provides a niche for mesenchymal stem cells of craniofacial bones.</a> Nat Cell Biol. 17 (4), 386-396 (2015).</li><li>Maruyama, T., Jeong, J., Sheu, T. -. J., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+of+the+suture+mesenchyme+in+craniofacial+bone+development,+repair+and+regeneration.">Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration.</a> Nat Commun. 7 (1), 10526 (2016).</li><li>Wilk, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+calvarial+skeletal+stem+cells+expressing+PRX1+reside+exclusively+in+the+calvarial+sutures+and+are+required+for+bone+regeneration.">Postnatal calvarial skeletal stem cells expressing PRX1 reside exclusively in the calvarial sutures and are required for bone regeneration.</a> Stem Cell Reports. 8 (4), 933-946 (2017).</li><li>Doro, D. H., Grigoriadis, A. E., Liu, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calvarial+suture-derived+stem+cells+and+their+contribution+to+cranial+bone+repair.">Calvarial suture-derived stem cells and their contribution to cranial bone repair.</a> Front Physiol. 8, 956 (2017).</li><li>Kajdic, N., Spazzapan, P., Velnar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniosynostosis-recognition,+clinical+characteristics,+and+treatment.">Craniosynostosis-recognition, clinical characteristics, and treatment.</a> Bosn J Basic Med Sci. 18 (2), 110-116 (2018).</li><li>Park, S., Zhao, H., Urata, M., Chai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sutures+possess+strong+regenerative+capacity+for+calvarial+bone+injury.">Sutures possess strong regenerative capacity for calvarial bone injury.</a> Stem Cells Dev. 25 (23), 1801-1807 (2016).</li><li>Quarto, N., Behr, B., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+spectrum+of+activity+of+canonical+Wnt+signaling+in+the+osteogenic+context+of+undifferentiated+and+differentiated+mesenchymal+cells:+Implications+for+tissue+engineering.">Opposite spectrum of activity of canonical Wnt signaling in the osteogenic context of undifferentiated and differentiated mesenchymal cells: Implications for tissue engineering.</a> Tissue Eng Part A. 16 (10), 3185-3197 (2010).</li><li>Kaku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cell-Induced+Cranial+Suture-Like+Gap+in+Rats.">Mesenchymal Stem Cell-Induced Cranial Suture-Like Gap in Rats.</a> Plast Reconstr Surg. 127 (1), 69-77 (2011).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+suture+regeneration+mitigates+skull+and+neurocognitive+defects+in+craniosynostosis.">Cranial suture regeneration mitigates skull and neurocognitive defects in craniosynostosis.</a> Cell. 184 (1), 243-256 (2021).</li><li>Koons, G. L., Diba, M., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+design+for+bone-tissue+engineering.">Materials design for bone-tissue engineering.</a> Nat Rev Mater. 5 (8), 584-603 (2020).</li><li>Mardas, N., Kostopoulos, L., Karring, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+and+suture+regeneration+in+calvarial+defects+by+e-PTFE-membranes+and+demineralized+bone+matrix+and+the+impact+on+calvarial+growth:+an+experimental+study+in+the+rat.">Bone and suture regeneration in calvarial defects by e-PTFE-membranes and demineralized bone matrix and the impact on calvarial growth: an experimental study in the rat.</a> J Craniofac Surg. 13 (3), 453-462 (2002).</li><li>Kostopoulos, L., Karring, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+sagittal+suture+by+GTR+and+its+impact+on+growth+of+the+cranial+vault.">Regeneration of the sagittal suture by GTR and its impact on growth of the cranial vault.</a> J Craniofac Surg. 11 (6), 553-561 (2000).</li><li>Mosaddad, S. A., Hussain, A., Tebyaniyan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+use+of+animal+models+in+craniofacial+regenerative+medicine:+A+narrative+review.">Exploring the use of animal models in craniofacial regenerative medicine: A narrative review.</a> Tissue Eng Part B Rev. , (2023).</li><li>Tan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PgC3Mg+metal-organic+cages+functionalized+hydrogels+with+enhanced+bioactive+and+ROS+scavenging+capabilities+for+accelerated+bone+regeneration.">PgC3Mg metal-organic cages functionalized hydrogels with enhanced bioactive and ROS scavenging capabilities for accelerated bone regeneration.</a> J Mater Chem B. 10 (28), 5375-5387 (2022).</li><li>Yazdanian, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+and+properties+of+&#946;TCP/Zeolite/Gelatin+scaffold+as+developed+scaffold+in+bone+regeneration:+in+vitro+and+in+vivo+studies.">Fabrication and properties of &#946;TCP/Zeolite/Gelatin scaffold as developed scaffold in bone regeneration: in vitro and in vivo studies.</a> Biocybern Biomed Eng. 40 (4), 1626-1637 (2020).</li><li>Soufdoost, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+evaluation+of+novel+Tadalafil/&#946;-TCP/Collagen+scaffold+for+bone+regeneration:+A+rabbit+critical-size+calvarial+defect+study.">In vitro and in vivo evaluation of novel Tadalafil/&#946;-TCP/Collagen scaffold for bone regeneration: A rabbit critical-size calvarial defect study.</a> Biocybern Biomed Eng. 39 (3), 789-796 (2019).</li><li>Russell, W. P., Russell, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy,+head+and+neck,+coronal+suture.">Anatomy, head and neck, coronal suture.</a> StatPearls. , (2022).</li><li>Menon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+stem+and+progenitor+cells+maintain+cranial+suture+patency+and+prevent+craniosynostosis.">Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis.</a> Nat Commun. 12 (1), 4640 (2021).</li></ol>

Conventional calvarial defect models, whether involving cranial sutures or not, primarily concentrate on the repair of hard tissue, often neglecting the vital regeneration of suture mesenchyme19,20. In suture regeneration research, prior models, like those by Mardas et al.15,16, utilizing a trephine bur to create a 5 mm circular defect across the sagittal suture of rats, resulted in substantial hard tissu...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Iodophor solution</td>
			<td>Chengdu Jinshan Chemical Reagent Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>75% Ethanol</td>
			<td>Chengdu Jinshan Chemical Reagent Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton balls</td>
			<td>Haishi Hainuo Group Co., Ltd.&#160;</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Lakong Medical Devices Co.,&#160;</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Chengdu Shifeng Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Dataviewer and Ctan software for residual defect volume assessments</td>
			<td>Bruker</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental low-speed round burs</td>
			<td>Dreybird Medical Equipment Co., Ltd.</td>
			<td>RA3-012 
			RA1-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile scalpel</td>
			<td>Hangzhou Huawei Medical Supplies Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringes (22 G)</td>
			<td>Chengdu Shifeng Co., Ltd.</td>
			<td>SB1-089(IX)</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>JASE</td>
			<td>BM320210</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>BioFroxx</td>
			<td>1340GR500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Stain Kit</td>
			<td>Biosharp</td>
			<td>BL700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Irrigation needle (23 G)</td>
			<td>Sichuan New Century Medical Polymer Products Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-speed handpiece</td>
			<td>Guangzhou Dental Guard Technology Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#8217;s Trichrome Stain Kit</td>
			<td>Solarbio</td>
			<td>G1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical non-woven fabrics</td>
			<td>Henan Yadu Industrial Co., Ltd.&#160;</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-computed tomography (&#181;CT)&#160;</td>
			<td>Scanco Medical AG</td>
			<td>&#181;CT45</td>
			<td></td>
		</tr>
		<tr>
			<td>Mimics 20.0 for cross-sectional images</td>
			<td>Materialise</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holders</td>
			<td>Chengdu Shifeng Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Periosteal elevator</td>
			<td>Chengdu Shifeng Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>Sichuan Kelun Pharmaceutical Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanco medical visualizer software for 3D image reconstruction</td>
			<td>Scanco Medical AG</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistics 20.0 for statistical analysis</td>
			<td>IBM</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats&#160;</td>
			<td>Byrness Weil Biotech Ltd</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Scissors</td>
			<td>Chengdu Shifeng Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Motor</td>
			<td>MARATHON</td>
			<td>N3-140232</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures (3-0 monofilament)</td>
			<td>Hangzhou Huawei Medical Supplies Co., Ltd.</td>
			<td>None</td>
			<td></td>
		</tr>
	</tbody>
</table>

the establishment of calvarial suture-bony composite defects in rats: a standardized model for suture-regenerative therapy investigation

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.

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.

In this study, the rat calvarial suture-bony composite defect was established by drilling a 4.5 mm x 2 mm rectangular hole across the coronal suture. The surgical schematic illustration and the research flow chart are depicted in Figure 3. The 3D image and the cross-sectional view of postoperative 0-day samples, namely samples collected immediately after surgery, confirmed the successful creation of a full-thickness calvarial defect, involving the complete removal of the coronal suture as we...

Read this Scientific Article Protocol about The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation at JoVE.com

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 ...

the-establishment-calvarial-suture-bony-composite-defects-rats