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 Figure 1A, including curved forceps, a disposable sterile scalpel, a periosteal elevator, an irrigation needle, cotton balls, a low-speed handpiece, a surgical motor, dental low-speed round burs (Figure 1B, 1.2 mm and 0.8 mm diameter, respectively), a needle holder, 3-0 monofilament sutures, and a straight scissor.</li>
	</ol>
	</li>
	<li>Sterilization and disinfection
	<ol>
		<li>Sterilize the instruments by steam sterilization (125-135 &#176;C, 20-25 min) in advance.</li>
		<li>Use ethanol to sterilize heat-sensitive medical equipment, such as electric shavers.</li>
		<li>Use sterile medical non-woven fabrics to cover the operation platform and disinfect the surrounding environment with 75% ethanol.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Prepare animals for surgery with a 1-week acclimation period.</li>
		<li>Inject the rats intraperitoneally with xylazine (10 mg/kg) and ketamine (100 mg/kg) 20 min before surgery for anesthesia or utilize any suitable anesthetic protocol to achieve general anesthesia. 
		NOTE: The ketamine-xylazine anesthetic cocktail administered intraperitoneally in rats typically takes effect within 10-15 min, reaching peak anesthesia around 35-40 min after injection. To prevent hypothermia during anesthesia, rats are best placed on a heating pad.</li>
		<li>Administer a preoperative subcutaneous injection of carprofen at 5 mg/kg to provide analgesia.</li>
		<li>Use the &#34;toe pinch method&#34; to determine whether or not the rats are conscious and responsive.</li>
	</ol>
	</li>
</ol>
2. Surgical process
<ol>
	<li>Site preparation
	<ol>
		<li>Place the rat in a prone position with its head naturally extending vertically, without requiring special devices or restraints to maintain this posture (Figure 2A).</li>
		<li>Apply veterinary ointment, i.e., Vaseline, to the rat&#39;s eyes to prevent corneal dryness.</li>
		<li>Remove the hair from the scalp between the nasal bridge and the cervical spine joint with an electric shaver (Figure 2B). Disinfect the surgical area in circular motions radiating from the center with 2% iodophor solution followed by 75% ethanol. Employ a surgical drape to cover the dorsum of the animal and the fur surrounding the incision.</li>
	</ol>
	</li>
	<li>Surgical site opening
	<ol>
		<li>Starting from the midpoint of the nasal bone, make a 2 cm longitudinal skin incision with a disposable scalpel following the midline of the cranium (Figure 2C).</li>
		<li>Make a midline periosteal incision mirroring the initial point and extent of the skin layer with a&#160;scalpel (Figure 2D). Then, gently lift the periosteum on both sides of the incision with a periosteal elevator (Figure 2E1) to expose parietal bones, frontal bones, and coronal sutures (Figure 2E2). 
		NOTE: When incising the periosteum, take care not to harm cranial sutures to prevent excessive bleeding when cutting. Adequate exposure of the coronal suture is of utmost importance for the following procedures.</li>
		<li>Rinse the wound with saline solution and dry the surgical area with cotton balls.</li>
	</ol>
	</li>
	<li>Suture-bony composite defect model establishment
	<ol>
		<li>Set the surgical motor to 35,000 rpm by rotating the knob (Figure 1A, yellow arrow), then turn on the switch (Figure 1A, white arrow).</li>
		<li>Starting from any point on the coronal suture, apply vertical force using a 1.2 mm diameter round bur until a sense of breakthrough is felt. 
		NOTE: Recommend the midpoint of the coronal suture (indicated by yellow arrows in Figure 2E2) as the starting point for penetration. When grinding, dental drills should be kept perpendicular to the cranial surface. Exercise caution not to continue drilling after penetrating the full thickness of the skull to prevent any further harm to the rats, including brain damage or cerebral hemorrhage.</li>
		<li>From the penetration point, move the bur laterally along the coronal suture to create an approximately 4 mm long positioning groove (Figure 2F1). Remove bone tissue with the bur on both sides of the positioning groove to initially form a rectangular full-thickness defect (Figure 2F2).</li>
		<li>Employ a 0.8 mm diameter round bur to refine details (Figure 2G1), involving the grinding of right angles and the smoothing of defect margins, ultimately achieving a standard rectangular defect measuring 2 mm in width and 4.5 mm in length (Figure 2G2). 
		NOTE: To achieve complete removal of the coronal suture while preserving the sagittal and frontal sutures in 300 g SD rats, the maximum feasible defect length is approximately 4.5 mm. Considering the width of the coronal suture in the anterior-posterior direction (Supplementary Figure S1), the defect width was set as 2 mm.</li>
		<li>Create two defects across the left and right halves of the coronal suture for self-comparison.</li>
		<li>Maintain continuous irrigation of saline solution during the drilling procedure to safeguard against thermal injury to the cranium and brain. Meanwhile, use cotton balls to dry the operation area.</li>
	</ol>
	</li>
	<li>Sample dimension verification
	<ol>
		<li>Regularly verify the length and width of the defects using a vernier caliper (Figure 2H1, H2) to ensure consistency across all samples.</li>
	</ol>
	</li>
	<li>Surgical site closure
	<ol>
		<li>Close the skin with 3-0 monofilament sutures (Figure 2I).</li>
	</ol>
	</li>
	<li>Immediate postoperative in vivo micro-computed tomography (&#181;CT)
	<ol>
		<li>If feasible, conduct in vivo &#181;CT scans on all rats immediately after surgery to confirm the success of the surgical procedure and monitor defect recovery trends for each individual.</li>
	</ol>
	</li>
</ol>
3. Postsurgical care
<ol>
	<li>According to animal care protocols, administer established analgesics as necessary after surgery, for instance, carprofen (5 mg/kg,&#160;subcutaneous use).</li>
	<li>Transfer the rats to a constant heating pad (37 &#176;C) for postoperative recovery.</li>
	<li>Once fully conscious, relocate the rats to their housing cage containing clean bedding. 
	NOTE: Continuously monitor the rats post-surgery. Do not leave them unattended until they can maintain sternal recumbency. Keep operated rats isolated from others until fully recovered.</li>
	<li>Conduct analgesia management and postoperative monitoring for 24 h, followed by daily assessments throughout the first week post surgery. Monitor the rats at least 1-2x per week thereafter.</li>
</ol>
4. Sample collection and data analysis
<ol>
	<li>Sample preparation
	<ol>
		<li>Collect cranium specimens at postoperative day 0, week 2, week 6, and week 12. Euthanize the rats using CO2 inhalation.</li>
		<li>Fix the samples in 4% paraformaldehyde at 4 &#176;C for 24 h before further analysis.</li>
	</ol>
	</li>
	<li>&#181;CT evaluation
	<ol>
		<li>Perform &#181;CT scans on cranial bones of postoperative day 0, week 6, week 12 with the following scan parameters: X-ray tube potential, 70 kVp; X-ray intensity, 0.2 mA; filter, AL 0.5 mm; integration time, 1 x 300 ms; and voxel size, 10 &#181;m.</li>
		<li>Obtain 3D reconstruction and cross-sectional images with image processing software (see Table of Materials).</li>
		<li>Measure residual defect volume and perform statistical analysis with corresponding software (see Table of Materials).</li>
	</ol>
	</li>
	<li>Histological staining
	<ol>
		<li>Decalcify the cranial bones in 12% (w/v) ethylene diamine tetraacetic acid solution (pH = 7.2) at 4 &#176;C for 6 weeks. 
		NOTE: Decalcify the samples with solution changes every 3 days. Utilize a shaker to expedite the process. Completion is indicated when a 25 G needle easily penetrates the sample.</li>
		<li>Proceed with dehydration, paraffin embedding, and preparation of 6 &#181;m sections using standard protocols18.</li>
		<li>Conduct histological analysis using hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining following kit protocol18.</li>
	</ol>
	</li>
</ol>

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>

The authors have no conflicts of interest to declare.

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.

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

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

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.

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 ...

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Research

JoVE Journal

Medicine

425 Views.  Sichuan University. 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. 

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

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Endotracheal Intubation Using a Flexible Intubation Endoscope as a Standardized Model for Safe Airway Management in Swine

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway or high ventilation pressures. Endotracheal intubation is a challenging skill,&#160;requiring a minimum number of successful endotracheal intubations to achieve a high success rate under optimal conditions, which is often unachievable for non-anaesthesiology researchers. Due to the specific porcine airway anatomy, a difficult airway can usually be assumed. The impossibility of establishing a secure airway can result in injury, adverse events, or death of the laboratory animal. Using a prospective, randomized, controlled evaluation approach, it has been shown that fiberoptic-assisted endotracheal intubation takes longer but has a higher first-pass success rate than conventional intubation without causing clinically relevant drops in oxygen saturation. This model presents a standardized regimen for endoscopically guided endotracheal intubation, providing a secured airway, especially for researchers who are inexperienced in the technique of endotracheal intubation via direct laryngoscopy. This procedure is expected to minimize animal suffering and unnecessary animal losses.

The use of pigs in research has increased in recent years. Nevertheless, pigs are characterized by difficult airway anatomy. By demonstrating how to perform endoscopically guided endotracheal intubation, the present protocol aims to further increase laboratory animals' safety to avoid animal suffering and unnecessary death.

Endotracheal intubation is often a basic requirement for translational research in porcine models for various interventions that require a secured airway ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...