The authors would like to thank Prof. Steven Nutt and Mr. Yuzheng Zhang for assistant with the Focus Ion Beam, and the Center for Electron Microscopy and Microanalysis (CEMMA) at USC for electron microscopy. Research was supported by NIH-NIDCR grants; DE-13414 and DE-020099 to J.M.O.

人类的臼齿提取以下在南加州大学牙奥斯特鲁夫学校的标准程序提取，并与机构审查委员会的批准处理。 1，酸腐蚀牙齿的制备切片<ol><li>选择一个人第三磨牙没有任何恢复龋齿。 </li><li>取出摩尔的根部，并切断摩尔的冠纵切成片2mm厚使用水冷式低速金刚石锯。超声波清洗这些片2分钟，然后用去离子水冲洗一下3倍。 </li><li>以模拟糜烂性病变，蚀刻牙片，用30％磷酸，持续30秒，然后立即冲洗它们与去离子水。 </li><li>超声波清洗蚀刻片2分钟，然后用去离子水冲洗一下3倍。 </li></ol> 2，釉原蛋白的制备壳聚糖水凝胶<ol><li>准备事项n个壳聚糖原液<ol><li>溶解1％（重量/体积），脱乙酰壳多糖（中分子量75-85％的脱乙酰化），在1％（V / V）的乙酸溶液中，随后在80℃的CO / N搅拌</li><li>将溶液冷却至室温后，用0.45μm的过滤器过滤脱乙酰壳多糖溶液。通过加入1M NaOH溶液将pH值调节至5.0。 </li></ol></li><li>钙与磷酸盐贮备液的制备（0.1M） <ol><li>称出氯化钙（ 氯化钙 ）和制备的0.1M溶液，然后混合使用涡旋直至溶液澄清。通过加入1M NaOH溶液将pH值调节到11。 </li><li>称取磷酸氢二钠（的Na 2 HPO 4）和制备的0.1M溶液，然后混合使用涡旋直至溶液澄清。 </li></ol></li><li>表达和重组釉原蛋白17-18的净化 这里所用的重组全长猪釉原蛋白（rP172）具有172个氨基酸，并且是充分的模拟长度的天然猪P173，但缺乏N-末端蛋氨酸，以及对Ser16 18上的磷酸基团。 <ol><li>加20克溶原性肉汤（LB）琼脂粉末到500ml去离子水，高压灭菌20分钟，将溶液在121℃（液体设置）。让琼脂冷却到〜55℃，加入氨苄青霉素（100微克/毫升）放入琼脂溶液。 </li><li>转移琼脂溶液至培养皿，让每个板冷却，直到它是固体，然后翻转，以避免在琼脂凝结。店板块在塑料袋在4°C </li><li>条纹的LB琼脂平板上以从甘油原液的重组细胞（ 大肠杆菌 -BC21）（-80℃）。孵育板O / N，37°C。 </li><li>准备50毫升NZCYM媒体和高压灭菌介质在121℃，30分钟。允许介质冷却，加50微升的100微克/毫升氨苄青霉素容量为50毫升的媒体。 </li><li>从LB琼脂平板上接种单菌落到辅以ampicill置50ml NZCYM媒体在37°C英寸孵育的细胞O / N在振荡培养箱</li><li>准备1升NZCYM媒体和高压灭菌介质在121℃，30分钟。加入1毫升100毫克/毫升的氨苄青霉素至千毫升媒体。采用了光学密度（OD）的阅读与使用紫外可见分光光度计的空白。注意：一读后不要丢弃此。 </li><li>接种从步骤2.3.5将10ml细胞培养物的成千毫升媒体从步骤2.3.6。接种后立即读出的光密度在595nm处。取的光密度读数定期跟踪生长的影响。注：外径取每次阅读的空白。 </li><li>诱导用700微升异丙醇-β-D-1-硫代半乳糖苷（IPTG，1M）的当细菌生长达到约0.75〜0.8 OD在595nm处。 </li><li>收获细胞，当外径达到1.1。倒入烧瓶中的内容复制到6的塑料瓶。平衡，并使用离心机前权衡瓶。离心20分钟，8,554×g离心，在4°C。保持细胞沉淀在-20℃的CO / N。 </li><li>取出细胞​​沉淀，并将其合并在一个50毫升管。添加3.5毫升4M盐酸胍的每升发酵。每次声处理2-3次，在冰上，30秒，以1分钟的间隔。 </li><li>使细胞体积与0.5％甲酸6倍稀释，让它在寒冷的房间2小时搅拌。离心30分钟，8,554 XG在4°C。保留上清液。加入20％饱和硫酸铵使用饱和原液，搅拌在寒冷的房间，O / N。 </li><li>离心30分钟，8554×g离心，在4℃，并保持颗粒。重建在0.1％三氟乙酸（TFA），负载粒料上的C4柱（10×250毫米，5微米），并分馏使用A = 0.1％TFA和B中的TFA = 60％乙腈的线性梯度洗脱，在一个流速1.5ml·分钟-1率。 </li><li>冻结干冰的蛋白质溶液和冻干的冷冻样品的O / N。 </li></ol></li><li>釉原蛋白，壳聚糖的制备（CS-AMEL）hydrogEL（ 图1A） <ol><li>加200 rp172微克到含有960微升的1％脱乙酰壳多糖溶液的管，然后混合使用涡旋直至溶液澄清。 </li><li>加25微升0.1M的氯化钙溶液中，接着加入15μl0.1M的磷酸氢二钠溶液与含釉原蛋白，脱乙酰壳多糖溶液，然后混合使用涡旋5分钟。 </li><li>通过仔细加入1M NaOH溶液调节CS-AMEL溶液至6.5的pH值。当pH值达到6.5的CS-AMEL水凝胶会形成。 </li></ol></li></ol> 3，搪瓷愈合中釉原蛋白，壳聚糖水凝胶<ol><li>人工唾液溶液的制备<ol><li>溶解一定量的氯化镁（MgCl 2的，0.2毫摩尔），氯化钙（ 氯化钙 ，1毫摩尔），磷酸二氢钾（KH 2 PO 4，4毫摩尔），氯化钾（氯化钾，16毫摩尔）和氯化铵（ 氯化铵 ，4.5毫摩尔）在20毫米肝素钠（4 - （2 -羟乙基）哌嗪-1 -乙烷磺酸）缓冲液中12。 </li><li>调节pH值至7.0 1 M氢氧化钠和人工唾液溶液储存在4℃ </li><li>使用该溶液前，加入氟化钠（NaF，为300ppm）。 </li></ol></li><li>的CS-AMEL水凝胶的应用<ol><li>应用约20微升CS-AMEL水凝胶，以使用注射器（ 图1B）经蚀刻的齿片。 </li><li>干燥的水凝胶覆盖的齿切片在干燥器中于室温搅拌2小时。 </li><li>牙片转移到含有30毫升人工唾液溶液的烧杯中。用铝箔覆盖烧杯中，然后保持该烧杯在烘箱中在37℃下7天。 </li><li>去除牙片和干燥在干燥器中于室温。 </li></ol></li></ol> 4，新生长层和搪瓷之间的接口特性的新生长层和搪瓷之间的界面的微观结构是安装前后通过扫描电子显微镜（SEM）和高转速透射电子显微镜（HR-TEM）粘弹性阻尼器。在TEM样品，用聚焦离子束（FIB）技术，为此，如下所示的步骤制备。 <ol><li>样品装入一个FIB-SEM的仪器，并根据操作指示完成所有的路线。注：细Ž调整应至少进行2次。 </li><li>沉积在样品上的碳层（15×3微米），以保护下面的结构（ 图2A）。 </li><li>磨小心将样品在碳层的上，下，右两侧，以制备薄片样品，然后切断该薄片的底侧。注：每个铣步骤（ 图2B）之前，请先检查离子束对准。 </li><li>在薄片焊接的Pt尖和剪切左侧分开的样品片（ 图3C）。注意：焊接步骤之前，请检查离子束对准。 </li><li>提起出吨他PT与样品尖慢，安装片状样品到电梯出TEM网格。 </li><li>分离来自样品的尖端，而薄的样品，直到它的厚度小于100纳米（ 图3D）。从仪器上取下样品进行TEM观察。注：每个细化的过程之前，请检查离子束对准。 </li></ol>新生长层的绑定和力学性能的影响5。评估<ol><li>的结合强度是通过超声波处理进行评估。保持牙齿修复片在超声波清洗机（42千赫，100 W）处理10分钟，然后观察修复层和天然牙釉质与背散射电子扫描电镜分析之间的接口。 </li><li>测量的硬度和弹性模量在每个修复牙釉质表面20个测试点（N = 3）由纳米压痕具有Berkovich压头。 </li></ol> 6，免疫荧光染色<ol><li>用Tris buffe洗齿片Ř盐水（TBS）15分钟。 </li><li>用1％牛血清白蛋白（BSA）在TBS中阻断15分钟。 </li><li>除去液体并孵育样品O / N 1°抗体（1:500鸡抗釉原蛋白）稀释在TBS（0.1％BSA，0.3％的Triton X-100）。 </li><li>样品用TBS 30分钟洗净，并培育O / N 2°抗体（1:100抗鸡FITC）稀释在TBS。 </li><li>样品用TBS 30分钟洗净，留下干燥30分钟。 </li><li>通过荧光显微镜观察。 </li></ol>

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The authors have nothing to disclose.

而牙釉质的矿物质含量高使得它在人体中最硬的矿化组织，这种生物陶瓷是易受除盐处理，这往往会出现龋齿或侵蚀。基因突变也可导致薄或软珐琅导致一系列的釉质畸形称为牙釉质发育不全20的遗传性疾病。含氟化物或CPP-ACP的口腔保健产品已在市场上几年（ 即 ，清漆，牙膏和漱口水），以促进初始釉质龋再矿化。然而，没有这些市售品，有促进组织磷灰石晶体的形成的潜...

Dental enamel is the hard mineralized surface of human teeth. It is composed of numerous needle-like apatite crystals, which are bundled in organized, parallel prisms to ensure the unique mechanical strength and biological protection that enamel provides1-2. Unlike other mineralized tissues, such as bone and dentin, mature enamel is acellular and cannot regenerate itself after substantial mineral loss1-2, which often occurs as dental caries or erosion. Commercially available products such as fluoride containing varnishes, tooth pastes and mouthwashes are effective in re-mineralizing enamel but none of them have the potential to promote the formation of organized apatite crystals. Clinically, the conventional treatment for enamel repair involves a filling procedure with artificial materials such as amalgam, ceramics, or composite resin 3. However, these materials usually do not interface well with the natural tissue surrounding the lesion, because their structures, components and properties are different from the natural enamel. As a result, secondary caries frequently develops overtime at the interface between the tooth and foreign materials. Therefore, in situ regrowth of enamel with a dense interface is an attractive target for the fields of materials science and stomatology. One particularly promising way to achieve this purpose is biomimetic synthesis of enamel-like material on the enamel surface. Recently, numerous in vitro attempts have been made to prepare enamel-like materials using biomimetic systems that contain nano-apatites or different organic additives 4-12. However, developing the optimal biomimetic strategy to promote remineralization crystals to achieve a perfect dense interface is still a challenge. 
During enamel mineralization, the oriented growth and elongation of apatite crystals is regulated by an amelogenin-rich matrix 1,13-14. To mimic the organic matrix in developing enamel, herein we describe a detailed protocol for fabricating an amelogenin-chitosan (CS-AMEL) hydrogel for in situ enamel regrowth on an acid-etched enamel surface used as a model for erosive lesions. As &#8220;the most versatile growth media&#8221; for crystals 15, hydrogel matrices have an advantage over a solution system since clinically they are easier to handle. Moreover, CS-AMEL is biocompatible, biodegradable, and has unique antimicrobial and adhesion properties that compare favorably with other biomimetic systems for dental applications 16. Importantly, the in situ mineralization of apatite crystals on the enamel surface provides a dense interface between the repaired layer and the natural enamel, which can potentially improve the durability of restorations and prevent the formation of new caries at the margin of the restoration.

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			<td style="width:164px;">Catalog Number</td>
			<td style="width:516px;">Comments/Description</td>
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			<td height="63" style="height:63px;width:331px;">Human Third Molar&#160;</td>
			<td style="width:209px;">Ostrow School of Dentistry of the University of Southern California&#160;</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">The human molars were extracted following the standard procedures for extraction at the Ostrow School of Dentistry of the University of Southern California and handled with the approval of the Institutional Review Board.</td>
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			<td height="42" style="height:42px;width:331px;">Recombinant Pocine Amelogenin</td>
			<td style="width:209px;">Expression and purification&#160; in lab</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">rP172, full-length&#160;</td>
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			<td height="21" style="height:21px;width:331px;">Chitosan&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">448877</td>
			<td style="width:516px;">medium molecular weight, 75-85% deacetylated</td>
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			<td height="21" style="height:21px;width:331px;">Phosphoric Acid&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA033266</td>
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			<td height="21" style="height:21px;width:331px;">Acetic Acid Glacial</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">A036289</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Hydroxide</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9292</td>
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			<td height="21" style="height:21px;width:331px;">Calcium Chloride&#160;</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:164px;">223506</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dibasic Sodium Phosphate Anhydrous</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0316</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">BL21-CodonPlus (DE3)-RP&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">230255</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Sulfate</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH8001</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Trifluoroacetic Acid</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAL06374</td>
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			<td height="21" style="height:21px;width:331px;">Acetonitrile</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH1103</td>
			<td style="width:516px;"></td>
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			<td height="21" style="height:21px;width:331px;">Magnesium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0244</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Dihydrogen Phosphate&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH9268</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Potassium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">BDH0258</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ammonium Chloride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAAA15000</td>
			<td style="width:516px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:331px;">HEPES (4-(2-Hydroxyethyl)piperazine-1-ethane-sulfonic acid)</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AAA14777</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sodium Fluoride&#160;</td>
			<td style="width:209px;">VWR</td>
			<td style="width:164px;">AA11561</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tris-Buffered Saline</td>
			<td style="width:209px;">Bio-Rad</td>
			<td style="width:164px;">170-6435</td>
			<td style="width:516px;">10&#215; TBS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Serum Albumin</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">12659</td>
			<td style="width:516px;">CalBioChem, Albumin, Bovine Serum, Fraction V, Low Heavy Metals&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Triton X-100</td>
			<td style="width:209px;">EMD Millipore&#160;</td>
			<td style="width:164px;">TX1568-1</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Chicken Anti-Amelogenin</td>
			<td style="width:209px;">N/A</td>
			<td style="width:164px;">N/A</td>
			<td style="width:516px;">A gift from Prof. Malcolm Snead, University of Southern California</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Bovine Anti-Chicken IgY-FITC</td>
			<td style="width:209px;">Santa Cruz Biotechnology</td>
			<td style="width:164px;">Sc-2700</td>
			<td style="width:516px;"></td>
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			<td height="32" style="height:32px;width:331px;">Equipments</td>
			<td style="width:209px;"></td>
			<td style="width:164px;"></td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:331px;">High Performance Liquid Chromatography System</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">Varian Prostar 210</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">C4 column</td>
			<td style="width:209px;">Phenomenx&#160;</td>
			<td style="width:164px;">Jupiter 5&#956; 300A</td>
			<td style="width:516px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Scanning Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JSM-7001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">FIB-SEM&#160;</td>
			<td style="width:209px;">JEOL&#160;</td>
			<td style="width:164px;">JIB-4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Transmission Electron Microscopy&#160;</td>
			<td style="width:209px;">JEOL</td>
			<td style="width:164px;">JEM-2100F</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital Low Speed Diamond Saw</td>
			<td style="width:209px;">MTI Corporation</td>
			<td style="width:164px;">SYJ-150</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Fluorescence Microscopy</td>
			<td style="width:209px;">Leica</td>
			<td style="width:164px;">DMI3000 B</td>
			<td style="width:516px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultrasonic Cleaner&#160;</td>
			<td style="width:209px;">Branson&#160;</td>
			<td style="width:164px;">2510</td>
			<td style="width:516px;">42 kHz, 100 W</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nano-indenter&#160;</td>
			<td style="width:209px;">Agilent Technologies Inc.</td>
			<td style="width:164px;">MTS XP</td>
			<td style="width:516px;"></td>
		</tr>
	</tbody>
</table>

development of amelogenin-chitosan hydrogel for in vitro enamel regrowth with a dense interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

这里描述的协议的有效性证明用扫描电子显微镜（SEM），选择区域电子衍射（SAED）和X-射线衍射（XRD）分析。修复由牙釉质蛋白 - 壳聚糖（CS-AMEL）水凝胶7天之后，形成在蚀刻釉质表面的搪瓷状层的厚度为15微米的。新生长层呈结晶的高度有序阵列的直径为约50 nm，优选沿c轴生长，垂直于表面（ 图3A中箭头）。值得注意的是，这些针状晶体被组织成束，这是类似天然牙釉质（ 图3...

Watch this Scientific Journal Video about Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface at JoVE.com

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

在这篇文章中，我们描述了一个协议，用于制造釉原蛋白，壳聚糖水凝胶浅表性搪瓷重建。组织在磷灰石晶体在水凝胶原位生长形成致密的搪瓷恢复界面，这将提高修复体的有效性和持久性。

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

development-amelogenin-chitosan-hydrogel-for-vitro-enamel-regrowth

Research

JoVE Journal

Bioengineering

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

14.9K 次观看.  University of Southern California. 在这篇文章中，我们描述了一个协议，用于制造釉原蛋白，壳聚糖水凝胶浅表性搪瓷重建。组织在磷灰石晶体在水凝胶原位生长形成致密的搪瓷恢复界面，这将提高修复体的有效性和持久性。 