This work is supported by National Natural Science Foundation of China 81825005 (L.Y.), 82201045 (F.Y.), and 82100982 (F.L.), and by Sichuan Province Science and Technology Program 2021JDRC0144 (F.L.), 2022JDRC0130 (F.Y.).

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-2017-041). Adult C57BL/6 mice, obtained from a commercial source (see Table of Materials), were used for the present study.
1. Presurgical preparation
<ol>
	<li>Instrument preparation
	<ol>
		<li>Prepare various needles of disposable syringes (26G, 25G, 23G; see Table of Materials) to ...

<ol>
	<li>Mamoun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+socket+etiology,+diagnosis,+and+clinical+treatment+techniques.">Dry socket etiology, diagnosis, and clinical treatment techniques.</a> Journal of the Korean Association of Oral and Maxillofacial Surgeons. 44 (2), 52-58 (2018).</li><li>Laraki, M., Chbicheb, S., El Wady, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolitis:+review+of+the+literature.">Alveolitis: review of the literature.</a> Odonto-Stomatologie Tropicale = Tropical Dental Journal. 35 (139), 19-25 (2012).</li><li>Soundia, A., 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=Osteonecrosis+of+the+jaws+(ONJ)+in+mice+after+extraction+of+teeth+with+periradicular+disease.">Osteonecrosis of the jaws (ONJ) in mice after extraction of teeth with periradicular disease.</a> Bone. 90, 133-141 (2016).</li><li>Ara&#250;jo, M. G., Silva, C. O., Misawa, M., Sukekava, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+socket+healing:+what+can+we+learn.">Alveolar socket healing: what can we learn.</a> Periodontology 2000. 68 (1), 122-134 (2015).</li><li>Hojo, H., Ohba, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sp7+Action+in+the+skeleton:+its+mode+of+action,+functions,+and+relevance+to+skeletal+diseases.">Sp7 Action in the skeleton: its mode of action, functions, and relevance to skeletal diseases.</a> International Journal of Molecular Sciences. 23 (10), 5647 (2022).</li><li>Zhou, 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=Multiple+functions+of+Osterix+are+required+for+bone+growth+and+homeostasis+in+postnatal+mice.">Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice.</a> Proceedings of the National Academy of Sciences. 107 (29), 12919-12924 (2010).</li><li>Ito, 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=Pathological+differences+in+the+bone+healing+processes+between+tooth+extraction+socket+and+femoral+fracture.">Pathological differences in the bone healing processes between tooth extraction socket and femoral fracture.</a> Bone Reports. 16, 101522 (2022).</li><li>Vasak, C., 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=Early+bone+apposition+to+hydrophilic+and+hydrophobic+titanium+implant+surfaces:+a+histologic+and+histomorphometric+study+in+minipigs.">Early bone apposition to hydrophilic and hydrophobic titanium implant surfaces: a histologic and histomorphometric study in minipigs.</a> Clinical Oral Implants Research. 25 (12), 1378-1385 (2014).</li><li>Ara&#250;jo, M. G., Lindhe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensional+ridge+alterations+following+tooth+extraction.+An+experimental+study+in+the+dog.">Dimensional ridge alterations following tooth extraction. An experimental study in the dog.</a> Journal of Clinical Periodontology. 32 (2), 212-218 (2005).</li><li>Kim, I. -. S., Ki, H. -. C., Lee, W., Kim, H., Park, J. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+systemically+administered+bisphosphonates+on+bony+healing+after+tooth+extraction+and+osseointegration+of+dental+implants+in+the+rabbit+maxilla.">The effect of systemically administered bisphosphonates on bony healing after tooth extraction and osseointegration of dental implants in the rabbit maxilla.</a> The International Journal of Oral &amp; Maxillofacial Implants. 28 (5), 1194-1200 (2013).</li><li>Kanyama, 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=Connective+tissue+growth+factor+expressed+in+rat+alveolar+bone+regeneration+sites+after+tooth+extraction.">Connective tissue growth factor expressed in rat alveolar bone regeneration sites after tooth extraction.</a> Archives of Oral Biology. 48 (10), 723-730 (2003).</li><li>Zhou, 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=The+role+of+IFT140+in+early+bone+healing+of+tooth+extraction+sockets.">The role of IFT140 in early bone healing of tooth extraction sockets.</a> Oral Diseases. 28 (4), 1188-1197 (2022).</li><li>Apaza Alccayhuaman, K. A., 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=FasL+is+required+for+osseous+healing+in+extraction+sockets+in+mice.">FasL is required for osseous healing in extraction sockets in mice.</a> Frontiers in Immunology. 12, 678873 (2021).</li><li>Avivi-Arber, L., Avivi, D., Perez, M., Arber, N., Shapira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+bone+healing+at+tooth+extraction+sites+in+CD24-deficient+mice:+A+pilot+study.">Impaired bone healing at tooth extraction sites in CD24-deficient mice: A pilot study.</a> PLoS One. 13 (2), 0191665 (2018).</li><li>Vieira, A. E., 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=Intramembranous+bone+healing+process+subsequent+to+tooth+extraction+in+mice:+micro-computed+tomography,+histomorphometric+and+molecular+characterization.">Intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization.</a> PLoS One. 10 (5), 0128021 (2015).</li><li>Min, K. -. 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=Effects+of+resveratrol+on+bone-healing+capacity+in+the+mouse+tooth+extraction+socket.">Effects of resveratrol on bone-healing capacity in the mouse tooth extraction socket.</a> Journal of Periodontal Research. 55 (2), 247-257 (2020).</li><li>Yu, F., Li, F., Zheng, L., Ye, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+controls+of+Sonic+hedgehog+guarantee+fidelity+of+epithelial+adult+stem+cells+trajectory+in+regeneration.">Epigenetic controls of Sonic hedgehog guarantee fidelity of epithelial adult stem cells trajectory in regeneration.</a> Science Advances. 8 (29), (2022).</li><li>Kuroshima, 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=Transplantation+of+noncultured+stromal+vascular+fraction+cells+of+adipose+tissue+ameliorates+osteonecrosis+of+the+jaw-like+lesions+in+mice.">Transplantation of noncultured stromal vascular fraction cells of adipose tissue ameliorates osteonecrosis of the jaw-like lesions in mice.</a> Journal of bone and Mineral Research. 33 (1), 154-166 (2018).</li><li>Ahel, V., 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=Forces+that+fracture+teeth+during+extraction+with+mandibular+premolar+and+maxillary+incisor+forceps.">Forces that fracture teeth during extraction with mandibular premolar and maxillary incisor forceps.</a> The British Journal of Oral &amp; Maxillofacial Surgery. 53 (10), 982-987 (2015).</li></ol>

The murine socket healing model is an important method for unraveling the underlying mechanisms in bone healing and regeneration, ultimately solving clinical challenges. Existing studies have demonstrated the possibility of the incisor extraction model and the maxillary molar extraction model, whereas studies have not used the mandibular first molar model13,17,18. However, incisors are crucial for rodent living, and their impair...

Socket healing after tooth extraction is a common clinical scenario, which can result in unbecoming complications such as socket hemorrhage, dry socket, or even jaw osteomyelitis under undesirable healing1,2,3. These comorbidities may impair the quality of life of patients and, even worse, vitally challenge prosthetic rehabilitation due to massive bone loss4. Though the socket healing stages have been elucidated, they are inadequate to direct clinical care post-tooth extraction surgery when encountering various prognosis challenges4.
Multiple studies based on animal models have been conducted to gain a better understanding of the underlying mechanisms in the socket healing process and avert the above situations. Sp7 is a master regulator in osteoblast differentiation, playing a vital role in skeleton development, bone hemostasis, and bone regeneration5,6. Rational socket healing models could display the redundancy of Sp7 post-traumatic in bone regeneration. In addition, distinct from long bone fracture healing, only a single osteogenic process, intramembranous ossification, involves the healing process of the extraction socket7. This makes the animal tooth extraction model optimal for studying implant-based therapies, as implant osseointegration obeys the same osteogenic rule8.
For decades, the tooth extraction model has been performed in rats, rabbits, and dogs, since these species have big teeth that are convenient to operate on9,10,11. However, given the flourishing demands for genetic modification and as a more adaptive genetic background to humans, mice are increasingly being used to establish a tooth extraction model. Thenceforward, researchers could unravel a specific cell population&#39;s role in the socket healing process using genome-modified mice instead of observing phenotypes only12. Among murine tooth extraction socket models, preceding studies have demonstrated the establishment and healing process of murine maxillary tooth and incisor extraction sockets13,14,15,16. However, the healing pattern of the prognosis, and the detective and observative time points, may differ across protocols. This appeals to a universal criterion for scholars to establish a murine socket healing model.
This study aimed to constitute a practicable murine socket healing model for the above issues. Mandible molars in mice have distinctive morphological traits compared to maxillary molars and incisors, bringing unique advantages and disadvantages. As the models focusing on the murine mandible are currently vacuum-based, this protocol tried to provide an accomplished method to extract the mandibular first molar in mice. We hope this protocol will enlighten basic researchers with new ideas to uncover underlying mechanisms of socket healing and indicate clinical care.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>23/25/26 G needle</td>
			<td>Chengdu Xinjin Shifeng Medical Apparatus &#38; Instruments Co. LTD.</td>
			<td>SB1-074(IV)</td>
			<td></td>
		</tr>
		<tr>
			<td>C57/B6J&#160;</td>
			<td>Gempharmatech Experimental Animals Company, Chengdu, China</td>
			<td>C57/B6J</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Staining Solution&#160;</td>
			<td>Beyotime&#160;</td>
			<td>Cat#C1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Cassettes</td>
			<td>CITOTEST Scientific</td>
			<td>80106-1100-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Stain Kit</td>
			<td>Biosharp</td>
			<td>BL700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#8217;s Trichrome Stain Kit</td>
			<td>Solarbio</td>
			<td>G1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome&#160;</td>
			<td>Leica</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital Sodium</td>
			<td>Huaxia Chemical Reagent Co., Ltd</td>
			<td>2018042001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal&#160;</td>
			<td>anti-Sp7&#160;</td>
			<td>Abcam Cat# ab22552</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Chengdu Xinjin Shifeng Medical Apparatus &#38; Instruments Co. LTD.</td>
			<td>SB2-115</td>
			<td></td>
		</tr>
	</tbody>
</table>

the establishment of a murine mandibular molar extraction socket healing model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

To elucidate the practical use of this method, the right mandibular first molar of two healthy C57BL/6 mice (3 months old, both female) were extracted and followed for 1 week and 4 weeks, respectively. The undamaged left mandibles were used as healthy controls. Figure 1A shows the specific features of the surgical appliance, including 26-23 G needles and a toothed ophthalmic tweezer. The 26 G needle is pinpoint-removed and bent. The 25 G needle is bent at approximately 25&#176; at the pinpoi...

Watch this Scientific Journal Video about The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model at JoVE.com

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone ...

This protocol provides a murine mandibular molar extraction model that is rarely introduced in available bone regeneration studies. Developing a murine mandibular molar extraction model is necessary as a mandible that is a more comparable and consistent among different intervention groups. The implications of this technique do not extend toward the therapy of any disease.However, it may help clinical healing and jaw bone regeneration in the long range by helping review the mechanisms underlying this process. This technique was developed to help the studies, revealing the mechanisms of available healing and regeneration. Regretfully, this method cannot be applied to other systems.Begin eliminating the distal resistance of the anesthetized mouse by holding the molar with tweezers medially. Then force a 23 gauge needle into the buccal alveolar bone of the distal root and render an interval. Next, change to a 25 gauge needle to continue the interval expansion.Progress delicately toward the periapical area while slowly rotating the needle forward and langually to press the root out of the alveolar fossa. To eliminate the mesial resistance, insert a 23 gauge needle into the root fork and lift the molar of occlusally. After holding the molar tightly take another 23 gauge needle and force it into the lingual mesial periodontal membrane to create an interval.Then use a 25 gauge needle and slowly rotate forward and buccally. If some underlying hindrances impede the luxation of the molar, use a 26 gauge needle to penetrate the root apex and repeat the operations. During the final extraction, extract the tooth ensuring that the crown rises above the occlusal plane and that two intact roots can clearly be seen.After successfully extracting the mandibular first molar, apply dry cotton to stop the bleeding, reposition the tongue, administer analgesia, and put the mouse on a constant temperature heating pad until it recovers from anesthesia. The tooth socket immediately filled with clot post extraction. The healing process of the socket was observed for one week.Some sponge-like trabecular bones were formed but the clot remained. In the healing process, the socket was filled with sponge bones after two weeks indicating the completion of regeneration. A master regulator in osteoblast differentiations Sp7 was widely expressed in the bone marrow cells in the margin which is the bone forming front.Under the homeostasis state the trabecular bones were consistent and confluent with bone marrow cell blocks sprinkled like islands. At one week post-surgery, numerous Sp7 expressing cells filled the extraction socket with newly formed trabecular bone sprinkled around. At four weeks post-surgery, the condition reversed and turned to largely merge to trabecular bone.The activity of Sp7 expressing cells declined to a level approaching the homeostasis state. The can also be performed to demonstrate the newborn regeneration process. This technique can help researchers unravel the underlying mechanisms of available healing and regeneration process by providing a new murine model.

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JoVE Journal

Biology

3.2K visualizações.  Sichuan University. Este protocolo fornece um modelo murino de extração de molares mandibulares que raramente é introduzido nos estudos de regeneração óssea disponíveis. O desenvolvimento de um modelo murino de extração de molares mandibulares é necessário como uma mandíbula mais comparável e consistente entre os diferentes grupos de intervenção. As implicações dessa técnica não se estendem para o tratamento de nenhuma doença.No entanto, pode ajudar a cicatrização clínica e regenera...