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 tissue loss, thereby straying from the primary goal of suture regeneration. Similarly, the methodology by Wilk et al.7, encompassing the removal of both the sagittal and right coronal sutures and creating a critical bone defect in the right parietal bone, although informative, introduced complexities and potential confounding variables due to the presence of multiple defect sites. In contrast, our protocol creates bilateral rectangular defects along the coronal suture, aiming for a more streamlined and targeted research model. The major cranial sutures include the coronal, sagittal, metopic, and lambdoid sutures21. Different from the metopic and sagittal sutures, which are longitudinally arranged on the skull, the coronal and lambdoid sutures are situated transversely and can be divided into two halves, facilitating the creation of two defects with identical conditions. In contrast to the lambdoid suture, the surgical field exposed by scalp incision is more accessible for a series of procedures at the coronal suture. Creating defects in both the left and right halves of the coronal suture and applying different interventions to each site facilitates a more straightforward comparison of distinct therapies through self-control. Moreover, this approach allows for a comparison between treated and untreated sides, providing insights into the disparities between the intervened healing process and the natural healing process. Therefore, our study suggests that the creation of suture-bony composite defects on both sides of the coronal sutures in rats is a suitable surgical model for studying suture-regenerative therapies.
The selection of defect dimensions is meticulously guided by practical and anatomical considerations. For this specific animal model, the primary challenge lies in completely removing the coronal suture while preserving the sagittal and frontal sutures, with minimal osseous tissue removal. The optimal strategy, therefore, entails the creation of a precise rectangular defect along the coronal suture. This strategic decision is indispensable, as any remaining mesenchyme in the coronal suture or adjacent sutures could introduce inaccuracies in evaluating the true regenerative impact of the treatment. Consequently, we established the largest feasible defect length for 300 g male SD rats at approximately 4.5 mm. Furthermore, taking into account the coronal suture&#39;s width in the anterior-posterior direction (approximately 1.5 mm in the sagittal plane, as shown in Supplementary Figure S1), we set the defect width at 2 mm to ensure the complete removal of the suture from the cranial surface to its depth. It is worth noting that the specific dimensions of the defect, both in length and width, may require adjustment based on factors such as the type and age of the animal, as well as the experimental objectives. Another study created a slender, 0.3-0.4 mm wide, rectangular defect along the coronal suture, accommodating the smaller size of mice13.
Notably, the difficulty in generating this surgical model lies in constructing standard and uniform rectangular defects. It is imperative to maintain consistency in the defect shape to guarantee a uniform dosage of bioactive factors across samples, thereby minimizing both inter-group and intra-group variations. In this regard, during the process of removing the suture-bony complex, we initially employed a 1.2 mm diameter round bur to locate the coronal suture and to create a rough outline of the full-thickness rectangular defect (Figure 1B). Subsequently, we switched to the smallest round bur (0.8 mm diameter) to refine the right-angle turns and smooth the defect edges (Figure 1B). To ensure the consistency of each defect&#39;s length and width, a vernier caliper was utilized to avoid over- or under-grinding during the drilling process. Despite this approach, the &#181;CT scan revealed that the rectangular defect we generated is still somewhat arc-shaped (Figure 4), suggesting the requirement for further optimization of the modeling method to obtain precise grinding shapes.
To evaluate the prognosis, &#181;CT scans provide the most direct means, including the assessment of bone tissue healing and the status of cranial sutures, whether they are closed or unobstructed. Moreover, investigation at the histopathological level is essential for the study of cranial suture regeneration. Histopathological staining helps in differentiating the non-closure of defects due to mesenchymal tissue formation, bone restorative inhibition, or material space-occupying. Only approaches that facilitate the regeneration of mesenchymal tissue are likely to possess the potential for cranial suture regeneration. Besides, despite the lack of relevant experiments during model construction, it is highly recommended to detect protein-level expression of suture MSC markers within the regenerated tissue following therapeutic interventions. This analysis, employing techniques such as immunofluorescence, immunohistochemistry, or flow cytometry, contributes to confirming the success of suture regeneration. Specific suture MSC markers include Cd51, Cd200, Gli1, Axin2, Prrx1, Ctsk, and more1,22.
In summary, this study reported the detailed modeling process of calvarial suture-bony composite defects in rats to study the prognosis of cross-suture calvarial defects and develop appropriate therapies for regenerating cranial sutures. It is pertinent to note that the field of cranial suture regeneration is indeed niche, with no standard models or methods prevalent in literature for regenerating cranial sutures. Thus, we believe that our protocol provides a solid methodological basis for investigating suture-regenerative approaches and introduces innovative perspectives on the functional restoration of calvarial defects, with the ultimate goal of enhancing patients&#39; quality of life.

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><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.&amp;nbsp;</td><td>None</td><td></td></tr><tr><td>Cotton swabs</td><td>Lakong Medical Devices Co.,&amp;nbsp;</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&lt;br/&gt; 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&amp;rsquo;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.&amp;nbsp;</td><td>None</td><td></td></tr><tr><td>Micro-computed tomography (&amp;micro;CT)&amp;nbsp;</td><td>Scanco Medical AG</td><td>&amp;micro;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&amp;nbsp;</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 well as the adjacent bone structures at both ends (Figure 4A). &#181;CT images and the corresponding statistical analysis results at 6 weeks post-operation revealed a natural inclination towards defect closure accompanied by irregular calcified nodules formed within the defect (Figure 4). This tendency became more pronounced 12 weeks after the surgery (Figure 4), indicating the potential suture fusion as time progressed. Further histological analysis was conducted using H&#38;E and Masson&#39;s trichrome staining. Two weeks postoperatively, we observed a remarkable extension and continuity of the periosteum and dura mater, which formed dense and thick fibrous tissue and effectively sealed the defect area (Figure 5). Notably, at this early time point, the emergence of blue-stained mineralized fibers was detected, whose presence underwent a substantial increase by the time of 6 weeks post-surgery (Figure 5). By postoperative 12 weeks, large pieces of new bone were observed in the defect center (Figure 5), reconfirming the adverse prognosis of stromal calcification and suture closure. The unfavorable natural healing outcomes of suture-bony composite defects emphasize the necessity of cranial suture regeneration through therapeutic interventions, such as the implantation of biomaterials13,15,16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66417/66417fig01v2.jpg" /> 
Figure 1: Surgical instruments. (A) The surgical items were numbered in order of use, including (1) curved forceps, (2) disposable sterile scalpel, (3) periosteal elevator, (4) irrigation needle, (5) cotton balls, (6) low-speed handpiece, (7) surgical motor (yellow arrow represents the RPM knob and white arrow represents the switch), (8) dental low-speed round burs, (9) needle holder, (10) 3-0 monofilament sutures, and (11) straight scissor. (B) Low-speed round burs of 1.2 mm and 0.8 mm diameters. <a href="https://www.jove.com/files/ftp_upload/66417/66417fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66417/66417fig02.jpg" /> 
Figure 2: Surgical procedure. (A) Position the rat in the prone position. (B) Shave the hair from the scalp with an electric shaver. (C) Incise skin with a disposable scalpel. (D) Incise the periosteum with a&#160;scalpel. (E1, E2) Elevate the periosteum to expose coronal sutures with a periosteal elevator. The yellow arrows indicate coronal sutures. (F1) Bore a positioning groove. (F2) Preliminarily create a rectangular defect with a 1.2 mm round bur. (G1, G2) Trim the defect with a 0.8 mm round bur to obtain a standard 4.5 x 2 mm rectangular shape. (H1, H2) Calibrate the width and length of the defect with a vernier caliper. (I) Close the skin using 3-0 monofilament sutures. This depiction utilizes a cadaver, which is deliberately left undraped to facilitate improved anatomical visualization. <a href="https://www.jove.com/files/ftp_upload/66417/66417fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66417/66417fig03.jpg" /> 
Figure 3: Schematic illustration of the calvarial suture-bony composite defect and research strategy of this study. Abbreviations: H&#38;E = hematoxylin and eosin; &#181;CT = micro-computed tomography. <a href="https://www.jove.com/files/ftp_upload/66417/66417fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66417/66417fig04.jpg" /> 
Figure 4: Closure tendency of the calvarial suture-bony composite defect. (A) Representative &#181;CT images and cross-sectional views of suture-bony composite defects at postoperative day 0, week 6, and week 12. The cross-sections depict the locations indicated by the yellow dashed lines in 3D images. Scale bars=2 mm. (B) Quantitative analysis of residual defect volume post-surgery through &#181;CT assessments. n = 6 replicates/group. Data are expressed as mean &#177; SD. One-way ANOVA followed by Dunnett&#39;s multiple comparisons test. ***p&#60;0.001. Abbreviations: PO = postoperative; &#181;CT = micro-computed tomography. <a href="https://www.jove.com/files/ftp_upload/66417/66417fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66417/66417fig05.jpg" /> 
Figure 5: Histological variations in suture-bony composite defects. (A) H&#38;E and (B) Masson&#39;s trichrome staining of suture-bony composite defects at&#160;PO&#160;week 2, week 6, and week 12. The red dashed outlines indicate the fibrous tissue encapsulating the defect area. The black pentagrams represent the blue-stained mineralized fibers. The yellow pentagrams highlight the new bone formed in the defect center. Scale bars = 500 &#181;m; 200 &#181;m (insets). Abbreviations: PO = postoperative; H&#38;E = hematoxylin and eosin. <a href="https://www.jove.com/files/ftp_upload/66417/66417fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Coronal suture width in the anterior-posterior direction (sagittal plane calvarial image). <a href="https://www.jove.com/files/ftp_upload/66417/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

450 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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengineering

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Developmental Biology

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

3D Visualization of Bone Remodeling Under Tensile Load in Suture Expansion Mice

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 facial bone growth, housing mesenchymal stem cells and osteoprogenitors. As most craniofacial bones develop through intramembranous ossification, the sutures' marginal regions act as initiation points. Due to this significance, these sutures have become intriguing targets in orthopedic therapies like spring-assisted cranial vault expansion, rapid maxillary expansion, and maxillary protraction. Under orthopedic tracing force, suture stem cells are rapidly activated, becoming a dynamic source for bone remodeling during expansion. Despite their importance, the physiological changes during bone remodeling periods remain poorly understood. Traditional sectioning methods, primarily in the sagittal direction, do not capture the comprehensive changes occurring throughout the entire suture. This study established a standard mouse model for sagittal suture expansion. To fully visualize bone remodeling changes post-suture expansion, the PEGASOS tissue clearing method was combined with whole-mount EdU staining and calcium chelating double labeling. This allowed the visualization of highly proliferating cells and new bone formation across the entire calvarial bones following expansion. This protocol offers a standardized suture expansion mouse model and a 3-D visualization method, shedding light on the mechanobiological changes in sutures and bone remodeling under tensile force loading.

Author Spotlight: PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling 

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force loading.

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...

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

Summary

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Chapters in this video

Use of Human Perivascular Stem Cells for Bone Regeneration

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

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