Name: 著者スポットライト:改良されたマウスモデルによる歯科歯髄研究の強化
Uploaded: 2023-10-27T13:30:00
Description: Watch this Scientific Journal Video about Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research at JoVE.com

この研究は、National Natural Science Foundation of China U21A20368 (L. Y.)、82101000 (H. W.)、82100982 (F. L.)、およびSichuan Science and Technology Program 2023NSFSC1499 (H. W.)からの助成金によって資金提供されました。この論文には、すべての元のデータと画像が含まれています。

この研究の実験手順は、四川大学西中国口腔病学院の倫理委員会(WCHSIRB-D-2021-125)によって承認されました。成体C57BL/6マウスは、Gempharmatech Experimental Animals Company(中国・成都)から入手しました。上顎第一大臼歯の冠全体は、生後21日で噴出します。手術を受けるマウスは、生後21日以上で、活力は正常であるべきである 11.ここでは、6週齢から8週齢のマウスをモデリングに用い?...

新規マウスギャグを用いた歯髄炎症マウスモデルの作成 

<ol>
	<li>Kleinert, A., Kleinert, L., Ozimirska, M., Cha&#322;as, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endodontium+-+together+or+separately.">Endodontium - together or separately.</a> Folia Morphol. 77 (3), 409-415 (2018).</li><li>Dhillon, H., Kaushik, M., Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+endodontics-Creating+new+horizons.">Regenerative endodontics-Creating new horizons.</a> J Biomed Mater Res B Appl Biomater. 104 (4), 676-685 (2016).</li><li>Prati, C., Pirani, C., Zamparini, F., Gatto, M. R., Gandolfi, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+endodontics:+a+review+of+current+status+and+a+call+for+action.">Regenerative endodontics: a review of current status and a call for action.</a> J Endod. 33 (4), 377-390 (2007).</li><li>Arora, 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=Potential+application+of+immunotherapy+for+modulation+of+pulp+inflammation:+opportunities+for+vital+pulp+treatment.">Potential application of immunotherapy for modulation of pulp inflammation: opportunities for vital pulp treatment.</a> Int Endod J. 54 (8), 1263-1274 (2021).</li><li>Eramo, S., Natali, A., Pinna, R., Milia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+regeneration+via+cell+homing.">Dental pulp regeneration via cell homing.</a> Int Endod J. 51 (4), 405-419 (2018).</li><li>Hasan, 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=Expression+of+Toll-like+receptor+2,+Dectin-1,+and+Osteopontin+in+murine+model+of+pulpitis.">Expression of Toll-like receptor 2, Dectin-1, and Osteopontin in murine model of pulpitis.</a> Clin Oral Investig. 27 (3), 1177-1192 (2023).</li><li>Wang, Y., 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=DDIT3+aggravates+pulpitis+by+modulating+M1+polarization+through+EGR1+in+macrophages.">DDIT3 aggravates pulpitis by modulating M1 polarization through EGR1 in macrophages.</a> Int Immunopharmacol. 120, 110328 (2023).</li><li>Richert, R., 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=A+critical+analysis+of+research+methods+and+experimental+models+to+study+pulpitis.">A critical analysis of research methods and experimental models to study pulpitis.</a> Int Endod J. 55 (Suppl 1), 14-36 (2022).</li><li>Huang, X. F., Zhao, Y. B., Zhang, F. M., Han, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+gene+expression+during+tooth+eruption+and+orthodontic+tooth+movement+in+mice.">Comparative study of gene expression during tooth eruption and orthodontic tooth movement in mice.</a> Oral Dis. 15 (8), 573-579 (2009).</li><li>Kwon, S. 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=Thermal+irritation+of+teeth+during+dental+treatment+procedures.">Thermal irritation of teeth during dental treatment procedures.</a> Restor Dent Endod. 38 (3), 105-112 (2013).</li><li>He, Y., 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=Pulpal+tissue+inflammatory+reactions+after+experimental+pulpal+exposure+in+mice.">Pulpal tissue inflammatory reactions after experimental pulpal exposure in mice.</a> J Endod. 43 (1), 90-95 (2017).</li><li>Karrar, R. N., 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=Molecular+biomarkers+for+objective+assessment+of+symptomatic+pulpitis:+A+systematic+review+and+meta-analysis.">Molecular biomarkers for objective assessment of symptomatic pulpitis: A systematic review and meta-analysis.</a> Int Endod J. 56 (10), 1160-1177 (2023).</li><li>Goldman, E., Reich, E., Abramovitz, I., Klutstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+apical+periodontitis+in+mice.">Inducing apical periodontitis in mice.</a> J Vis Exp. (150), e59521 (2019).</li><li>Duncan, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+status+and+future+directions-Vital+pulp+treatment+and+pulp+preservation+strategies.">Present status and future directions-Vital pulp treatment and pulp preservation strategies.</a> Int Endod J. 55 (Suppl 3), 497-511 (2022).</li><li>Shi, X., Li, Z., He, Y., Jiang, Q., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+dental+burs+for+experimental+induction+of+pulpitis+in+mice.">Effect of different dental burs for experimental induction of pulpitis in mice.</a> Arch Oral Biol. 83, 252-257 (2017).</li><li>Du, W., 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=Indigenous+microbiota+protects+development+of+medication-related+osteonecrosis+induced+by+periapical+disease+in+mice.">Indigenous microbiota protects development of medication-related osteonecrosis induced by periapical disease in mice.</a> Int J Oral Sci. 14 (1), 16 (2022).</li></ol>

歯髄は、歯の中の孤立した軟組織として、歯の生理活性を維持する上で重要な役割を果たしていますが、依然として非常に敏感です。この重要な歯髄の保存は、最近の歯内療法において好ましい初期アプローチとなっており、歯髄の炎症メカニズムを包括的に理解する必要があります16。炎症性微小環境の時空間変動と歯髄炎の常在細胞型間の相互作用は、 in vitro 研?...

歯の不可欠な部分である歯髄は、栄養供給、象牙質形成、感覚機能、防御反応など、複数の重要な機能を担っています1。それにもかかわらず、硬組織に囲まれた歯髄は、深部虫歯、歯髄炎、外傷、またはその後の治療による損傷や損傷を受けやすいです2,3。機能的な歯髄がないと、歯の脆弱性のリスクが高まります4。さらに、若い永久歯の歯髄の活力の喪失は歯の成熟に悪影響を与える可能性があり、現在の義歯技術は健康な歯髄によって提供される神経フィードバックを回復できません4。このような状況により、研究者は、単なる除去だけでなく、炎症を起こした歯髄を管理するための代替ソリューションを模索するようになりました。
2007年、Murrayらは再生歯内療法における組織工学の応用を開始し、それによって歯髄の保存と再生5への関心が高まりました。しかし、炎症を起こした歯髄組織は、細胞がIL-6などの炎症性因子を放出し、炎症細胞を動員し、細胞の壊死、歯髄の活力の喪失、および機能回復の合併症を引き起こすため、課題となります6,7。したがって、炎症とそれに伴う細胞死を理解することは、生命パルプの保存を進歩させるために非常に重要です。in vivoまたはin vitroで炎症果肉の分子生物学を探求するために実施された多くの実験があります8,9。2Dまたは3D細胞培養のようなin vitro実験は長年にわたって開発され、成熟しつつあり、炎症因子に対する歯髄細胞の反応をテストするために広く使用されつつあるが、これらの実験は歯髄組織と全身免疫系との間の相互作用を反映することはできない10。研究されている現象が免疫系、血管系、神経系などの他の組織起源の細胞に由来する場合、純粋な歯髄細胞培養は行き詰まりにつながります。したがって、in vivo実験は非常に必要であり、参考になります。
マウスは、その費用対効果、高い生殖能力、および活力により、 in vivoでの炎症研究においてますます一般的な選択肢になっています。ただし、マウスの歯髄炎モデルの包括的なプロトコルは現在存在しておらず、参照として役立ちます。マウスのサイズが小さく、刺激に対する感受性が高いため、実験手順では大きな課題が生じます。マウスの口の奥深くに隠された極小の歯を観察するには、実験室で卓上顕微鏡がより一般的に存在するにもかかわらず、カンチレバー顕微鏡の使用が必要になることがよくあります。開口器がないため、他の人の助けが必要です。これに対処するために、グループは、マウスの歯髄炎モデルを構築するための標準化された再現可能なプロトコルを提供することを目的とした、容易に入手できる材料を使用したマウスギャグを考案しました。この記事では、C57マウスの術前準備、固定化、歯髄曝露手術、およびサンプル採取を含む手順について詳しく説明します。このプロトコルでは、マウスギャグの使用を推奨し、その構造、製造、および適用に関する情報を提供して、他の研究者が手順を再現するのを容易にします。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57/B6J mice</td>
			<td>Gempharmatech Experimental Animals Company</td>
			<td>C57/B6J</td>
			<td>For the establishment of pulp exposure</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Chengdu Xinjin Shifeng Medical Apparatus &#38; Instruments Co. LTD.</td>
			<td>SB1-074(IV)</td>
			<td>Apply in drug injection.</td>
		</tr>
		<tr>
			<td>8# C+ file</td>
			<td>Readysteel</td>
			<td>0010047</td>
			<td>Apply in exposing the roof of pulp chamber.</td>
		</tr>
		<tr>
			<td>Anesthesia Mix solution</td>
			<td></td>
			<td></td>
			<td>10% ketamine hydrochloride+ 5% xylazine + 85% sterile isotonic saline.&#160;</td>
		</tr>
		<tr>
			<td>DAPI Staining Solution</td>
			<td>Beyotime</td>
			<td>C1005</td>
			<td>Apply in immunofluorescence staining for counter-staining of nucleus.</td>
		</tr>
		<tr>
			<td>Dental high-speed dental handpiece</td>
			<td>Jing yuan electronic commerce technology</td>
			<td>WJ-422</td>
			<td>Apply in pulp exposure.</td>
		</tr>
		<tr>
			<td>Heavy wire cutter</td>
			<td>Jirui Medical Instrument Co., Ltd.</td>
			<td>none</td>
			<td>Apply inarc cutting.</td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Stain kit</td>
			<td>Biosharp</td>
			<td>BL700B</td>
			<td>For the histological analysis of the slides.</td>
		</tr>
		<tr>
			<td>IL-6 antibody</td>
			<td>Novus</td>
			<td>NBP2-89149</td>
			<td>Apply in immunofluorescence staining to detect the inflammation of the dental pulp.</td>
		</tr>
		<tr>
			<td>Ketamine(Ketamine hydrochloride)</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>C3N VT1</td>
			<td>100mg/kg, IP. Apply in nesthetization.</td>
		</tr>
		<tr>
			<td>Medical tap</td>
			<td>3M</td>
			<td>1530</td>
			<td>Apply in mice immobilization.</td>
		</tr>
		<tr>
			<td>Orthodontic arch wire&#160;</td>
			<td>Shanghai Wei Rong Medical Apparatus Co. LTD.</td>
			<td>K417</td>
			<td>Diameter of 8&#181;m</td>
		</tr>
		<tr>
			<td>Round dental burr (0.6 mm)</td>
			<td>Shofu global</td>
			<td>072208</td>
			<td>Apply in removing enamel and shallow layer of dentin.</td>
		</tr>
		<tr>
			<td>Young loop bending plier</td>
			<td>Jirui Medical Instrument Co., Ltd.</td>
			<td>none</td>
			<td>Apply in arc bending.</td>
		</tr>
	</tbody>
</table>

establishment of a murine pulp exposure model with a novel mouth-gag for pulpitis research

歯髄炎は、自然な歯の喪失の一般的な原因であり、炎症を起こした歯髄の壊死と生理活性の喪失につながります。歯髄炎の根底にあるメカニズムとその効率的な治療を解明することは、歯内療法研究の継続的な焦点です。したがって、歯髄内の炎症過程を理解することは、歯髄の保存を改善するために不可欠です。他の in vitro 実験と比較して、マウス歯髄炎モデルは、歯髄炎の病理学的進行を観察するための、より本物で遺伝的に多様な状況を提供します。しかし、マウスの使用は、費用対効果が高く、アクセスしやすいにもかかわらず、サイズが小さく、協調性が低く、耐性が低いため困難を伴い、口腔内および歯科治療を複雑にします。このプロトコルは、マウスの髄を露出させるためのマウスギャグの新しい設計と適用を導入し、より効率的な口腔内処置を促進します。マウスギャグは、ほとんどの歯科医がすぐに利用できる歯列弓で構成されており、初めての手術でも手術の準備を大幅に迅速化できます。マイクロCT、ヘマトキシリン-エオシン(HE)染色、および免疫蛍光染色を使用して、形態と細胞発現の変化を特定しました。この記事の目的は、研究者がこの新しい口ギャグを使用して歯髄炎症モデルを作成するための、より再現性が高く、要求の少ない手順を確立するのを支援することです。

The dental pulp, an integral part of the tooth, is responsible for multiple essential functions such as nutrient supply, dentin formation, sensory function, and defense reactions1. Nevertheless, the dental pulp, surrounded by hard tissue, is susceptible to injuries and damages from deep caries, pulpitis, trauma, or subsequent therapies2,3. The absence of functional dental pulp increases the risk of tooth fragility4. Moreover, the loss of pulp vitality in young permanent teeth can adversely affect tooth maturation, and current denture techniques fail to restore the neural feedback offered by healthy pulp4. This situation has led researchers to explore alternative solutions for managing inflamed pulp beyond mere removal.
In 2007, Murray et al. initiated the application of tissue engineering in regenerative endodontics, thereby sparking increased interest in pulp preservation and regeneration5. However, inflamed pulp tissue poses a challenge as cells release inflammatory factors such as IL-6, which recruit inflammatory cells and results in cell necrosis, loss of pulp vitality, and complications in functional recovery6,7. Understanding inflammation and the associated cell death is, therefore, crucial for advancements in the preservation of vital pulp. There are a number of experiments that have been conducted to explore the molecular biology of the inflame pulp in vivo or in vitro8,9. Though in vitro&#160;experiments like 2D or 3D cell cultures have been developed for years and are becoming mature and widely used to test reactions of pulp cells to inflammatory factors, these experiments cannot reflect the interaction between pulp tissue and the systemic immune system10. If the phenomenon being studied is derived from cells of other tissue origin like immune, vascular and nervous system, then pure pulp cell culture will lead to a dead end. Therefore, in vivo experiments are very necessary and referential.
Mice have increasingly become a common choice in inflammation research in vivo&#160;due to their cost-effectiveness, high fertility, and vitality. However, a comprehensive protocol for mice pulpitis model is currently absent, which can serve as a reference. The small size of mice and their sensitivity to stimulation pose significant challenges during experimental procedures. Observing the minuscule teeth concealed deep within the mouse mouth often necessitates the use of a cantilever microscope, notwithstanding the more common presence of desktop microscopes in laboratories. The absence of a mouth opener requires assistance from others. To address this, the group has devised a mouth-gag using readily available materials which aims to provide a standardized and reproducible protocol for constructing the mice pulpitis model. This article details the procedure, covering preoperative preparation, immobilization, pulp exposure surgery, and sample collection on C57 mice. This protocol recommends the use of the mouth-gag, providing information on its structure, production, and application to facilitate other researchers in replicating the procedure.

著者スポットライト:改良されたマウスモデルによる歯科歯髄研究の強化

歯髄炎研究のための新規マウスギャグを用いたマウスパルプ曝露モデルの確立

Individual experiments like 2D or 3D cell cultures have been widely used to test the reactions of dental pulp cells. These experiments cannot reflect the interaction between pulp tissue and the systemic immune system. Therefore, in vivo experiments are becoming increasingly popular in endodontic research.C57 mice have increasingly become a common choice in inflammation research on dental pulp due to their cost effectiveness, high fertility and vitality. However, the small size of mice and lack of coordination posed significant challenges during experimental procedures. The fixation process is an important factor that higher the technical threshold.We devised a novel mouth gag using readily available materials and simple assemblies to assist researchers in pulp exposure surgery. According to our research, this mouth gag can significantly shorten the time required for beginners to immobilize the mouth and expose the maxillary first molar of mice.

上記の手順は、3、6〜8週齢のC57BL / 6マウスの右上顎第一大臼歯で行われましたが、左上顎第一大臼歯はコントロールとして保存されました。ブランクコントロール、12時間歯髄炎および24時間歯髄炎サンプルからの組織学および免疫蛍光結果をデモンストレーションに利用しました。
Goldman et al.15 の CT 解析のプロトコルに従って、図 4</str...

Watch this Scientific Journal Video about Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research at JoVE.com

この記事では、革新的なマウスギャグを使用してマウスの歯髄炎モデルを確立するための合理化されたプロトコルと、その後の組織学的分析について説明します。

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms ...

2Dまたは3D細胞培養などの個々の実験は、歯髄細胞の反応をテストするために広く使用されています。これらの実験は、歯髄組織と全身性免疫系との間の相互作用を反映することはできません。そのため、歯内療法の研究において、in vivo実験はますます人気が高まっています。C57マウスは、その費用対効果、高い繁殖力、活力により、歯髄の炎症研究においてますます一般的な選択肢となっています。しかし、マウスのサイズが小さく、協調性が欠如していることが、実験手順において大きな課題となっていました。固定プロセスは、技術的なしきい値を高くするための重要な要素です。私たちは、手軽に入手できる材料と簡単なアセンブリを使用して、研究者が歯髄曝露手術を行うのを支援するために、新しいマウスギャグを考案しました。私たちの研究によると、この口ギャグは、初心者がマウスの上顎第一大臼歯を露出させ、口を固定するのに必要な時間を大幅に短縮することができます。

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Medicine

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

医学

Preparation of the Mouth Gag for a Mouse Pulpitis Model

To begin, straighten an 8 micron thick orthodontic arch wire with your left hand while keeping the right hand slightly bent against the wire arc. Use a Young loop bending plier to bend the top edge of the trapezoid about eight millimeters at the midpoint of the bow wire. Ensure that point A is on the edge of the plier beak.Now hold the plier with the left hand. Clamp the free end of the bow wire with the right thumb and forefinger, then bend the bow wire from point A to make a 120-degree angle. Place the arch wire on a horizontal table to check if it is on a single plane.Leave about nine millimeters on each side of the wire and use pliers to bend the free end to a 75-degree angle. Now locate point C which is about five millimeters from point B.Bend the wire at point C to make a 105-degree angle. Similarly, bend the wire at points D and E.Clamp the IK and KJ parts simultaneously.Duplicate the A, B, C bending at points L, J, and K, then bend the free end at point J to make it vertical to the IKJ plane. Bend an extra tongue depressor for the mandibular part. Clamp point I five millimeters from point J and bend the free end to make it parallel to both the IKJ plane and the CD part.Next, leave a five millimeter gap from point I and at point H, bend the arch vertically to the IH and JIH planes. Locate point G five millimeters from H and clamp the JIHG lane. Then bend the arch at point G.Now bend the free end symmetrical to the KJ plane.The free end after point F should be symmetrical to the free end at point E.Finally, place rubber caps on the free ends.

マウス歯髄炎モデルのための口ギャグの準備

Using A Mouth Gag for Pulpitis Assessment in Mice

To begin, fix an anesthetized mouse in a supine position on a fixation plate. Secure the limbs with skin tape. Compress the free ends of a prepared mouth gag with the thumb and the index finger.Fix the mouse's front incisors in the trapezoidal groove between the two arms of the mouth gag. Ensure the arm with the tongue depressor is placed on the mandibula. Check the maxillary first molar to ensure it is free of dental caries, trauma, and odontogenesis.Assess the surrounding gingiva to ensure absence of redness, swelling, or fistula. Then, check the health of the opposite teeth. Next, with a dental burr, drill the occlusal side of the maxillary first molar at 20, 000 RPM.Remove the enamel. With a syringe, drop normal saline every three minutes over the tooth to prevent overheating. Put a number 8 C+file on the lowest position of the drilled pit and pierce the last dentine to expose the pulp chamber.Clean the fragments around the tooth, then remove the mouth gag. The time taken to fix the mouth with the mouth gag and to find the maxillary first molar was significantly shortened relative to the traditional method. The pulp exposure was confirmed through micro CT and reconstruction modeling.At 24 hours post-surgery, most of the pulp tissue retained its morphology.