CN112057205A - 一种用于牙槽骨位点保存的3d打印牙根支架及其制备方法 - Google Patents
一种用于牙槽骨位点保存的3d打印牙根支架及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于牙槽骨位点保存的3D打印牙根支架及其制备方法,所述用于牙槽骨位点保存的3D打印牙根支架为中空三维连通结构,主体材料为β‑磷酸三钙,表面粘附有乳牙牙髓干细胞并进行成骨诱导。本发明将生物陶瓷材料(β‑磷酸三钙)、干细胞、3D打印技术的优势相互结合,将其联合应用于位点保存术上,3D打印的TCP支架具有良好的生物学特性,并且乳牙牙髓干细胞可作为负载其上的良好骨组织工程种子细胞;乳牙牙髓干细胞联合TCP支架材料可有效保存拔牙后的牙槽骨水平宽度及牙槽嵴颊侧骨板垂直高度,并促进牙槽窝内新骨的形成。
Description
技术领域
本发明涉及医用支架技术领域,更具体地,涉及一种用于牙槽骨位点保存的3D打印牙根支架及其制备方法。
背景技术
临床上牙周病、根尖周病及外伤等因素常导致牙齿的丢失。牙槽骨的形状和体积由牙齿的存失、形状、生长方向和牙长轴决定,当牙齿丢失或拔除时,硬组织及软组织重塑的生理过程会导致牙槽嵴在高度和宽度上的减少,减少的程度与多种变量因素有关,包括牙槽窝大小、粘膜厚度、代谢因子和功能负荷。有研究分析表明,拔牙后6个月的水平骨丢失率为29%至63%,垂直骨丢失率为11%至22%。再吸收模式的特点是前3-6个月快速减少,随后逐渐减少。牙槽骨的吸收往往对后期的种植修复及美学造成不利的影响,如何有效地保存牙缺失后的牙槽骨的宽度及高度得到了现阶段种植及修复医生的高度重视。
牙槽骨是全身唯一有牙齿附着的骨,并且无时无刻不在发生着改建,牙槽骨的改建与牙齿密切相关。在这些改建的过程中,成骨细胞和破骨细胞维持着微妙的平衡。在咀嚼时,咬合力会通过牙齿周韧带传导到牙槽骨上。而牙齿缺失后,该处的牙槽骨由于失去了来自牙周膜的刺激,会发生生理性的骨吸收。临床上,这些吸收最终表现为牙槽嵴高度和宽度的显著减少。研究发现,牙齿缺失后3-12个月间牙槽嵴的宽度可丧失2.6-4.6mm,高度可丧失0.4-3.9mm,从拔牙开始3个月内骨吸收总量可达50%以上,而牙槽骨在唇侧骨板发生的吸收大于舌侧。牙齿缺失后发生的牙槽骨吸收会对口腔颌面部的健康造成危害,若牙槽嵴吸收过多,不仅会影响后期的种植及修复治疗,牙槽骨上附着牙龈的塌陷还会对患者的美观程度造成较大的影响。因此,为了尽量保留牙齿缺失后的牙槽骨骨量,位点保存术是必须的。目前研究认为,位点保存术的最佳时间为拔牙后即刻。
膜(membrane)在口腔医学领域中应用广泛,在牙槽嵴保存术上主要起引导骨组织再生(guide bone regeneration,GBR)作用。GBR屏障膜防止结缔组织及上皮组织长入骨缺损区,从而留出足够的空间给血液中的祖细胞及周围的骨细胞迁移入缺损区来形成新骨,GBR膜根据其降解特性分为不可吸收膜生物膜与可吸收膜生物膜。由于聚四氟乙烯等不可吸收的生物膜在体内不可降解,因此需要二次手术取出,这不仅会对患者造成二次创伤,且增加了创区发生感染的风险。组织补片、bio-Gide、Mucograft membrane、Mem-Lok Pliable等新型可吸收膜生物膜逐渐被用于位点保存。
但是,由于生物膜硬度不足,难以维持足够的牙槽骨空间,这限制了其临床使用,而骨替代材料可为其形成有效的支撑,从而获得足够的空间。目前应用于位点保存术的骨替代材料主要来自于自体骨、人造骨、异种骨和同种异体骨。
然而,位点保存术单独使用骨替代材料覆盖或不覆盖生物膜均不能有效阻止拔牙后的牙槽嵴吸收,也不能确保其能促进新骨形成(相反,材料的存在反而会阻碍拔牙创的正常愈合过程),目前的位点保存术还无法有效阻止牙槽嵴的吸收。
发明内容
本发明为克服上述现有技术所述的用于牙槽骨位点保存的骨替代材料和/或生物膜无法有效阻止牙槽嵴的吸收,也无法促进新骨形成的缺陷,提供一种用于牙槽骨位点保存的3D打印牙根支架,提供的3D打印牙根支架用于牙槽骨位点保存时,能够有效保存拔牙后的牙槽嵴水平宽度及牙槽嵴颊侧骨板垂直高度,并促进牙槽窝内新骨的形成。
本发明的另一目的在于提供上述3D打印牙根支架的制备方法。
为解决上述技术问题,本发明采用的技术方案是:
一种用于牙槽骨位点保存的3D打印牙根支架,所述3D打印牙根支架为中空三维连通结构,主体材料为β-磷酸三钙,表面粘附有乳牙牙髓干细胞并进行成骨诱导。
磷酸三钙(tricalcium phosphate,TCP)的物理、化学性能以及生物相容性与人正常骨组织中的羟基磷灰石十分相近,具有良好的骨传导性及可降解性,在降解时释放的钙离子与磷酸根离子可参与局部体液循环,并被周围骨组织利用,促进新骨形成。3D打印技术的出现可以将支架材料塑造为临床所需的个性化形态,并在其中塑造多孔结构来加强组织工程种子细胞的附着环境。
虽然颗粒型材料可以一定程度上对牙槽窝起到支撑作用,但其在形状可塑性以及需要修复的部位体积和空间维持的效果是不理想的,因此,通过3D打印技术制作成型牙根形态的骨替代材料有着广泛的应用前景。
乳牙牙髓干细胞(stem cells from human exfoliated deciduous teeth,SHED)具有高度增殖、多向分化潜能和自我更新等生物学特性。相对于骨髓间充质干细胞,SHED的增殖率更高,能为临床治疗提供足够数量的细胞。在成骨方面,SHED表达碱性磷酸酶(ALP)、I型胶原、骨粘连蛋白、骨桥蛋白(OPN)和骨钙素(OCN)等成骨相关标记物,在体外经矿化培养液诱导后,成骨标记物表达上调,茜素红染色有钙结节形成,证明其具有成骨分化和体外形成矿化组织的能力。在SHED移植至体内后向成骨分化的过程中,会表达高水平的OCN和ALP,从而诱导体内的成骨细胞来参与新骨形成,表现出很强的骨诱导能力。因此,其可在组织工程中为支架提供良好的骨诱导性及骨形成性,并且由于SHED在牙髓组织中位于血管周围,因此它还具有良好的成血管性能。
本发明将生物陶瓷材料(β-磷酸三钙)、干细胞、3D打印技术的优势相互结合,将其联合应用于位点保存术上,3D打印的TCP支架具有良好的生物学特性,并且乳牙牙髓干细胞可作为负载其上的良好骨组织工程种子细胞;乳牙牙髓干细胞联合TCP支架材料可有效保存拔牙后的牙槽骨水平宽度及牙槽嵴颊侧骨板垂直高度,并促进牙槽窝内新骨的形成。
优选地,所述3D打印牙根支架上乳牙牙髓干细胞的粘附量为105~7个/g。
优选地,所述3D打印牙根支架上乳牙牙髓干细胞的粘附量为106个/g。
优选地,所述3D打印牙根支架的侧壁孔径为50~100μm,横断面孔径为350~400μm,孔隙率为50%~60%。
本发明还保护上述3D打印牙根支架的制备方法,所述制备方法包括如下步骤:
S1.根据待保存位点的形状建立三维模型;
S2.配制主要含有β-磷酸三钙的打印浆料,通过3D打印机根据步骤S1的三维模型打印、烧结得到TCP支架;
S3.将步骤S2的TCP支架浸泡在乳牙牙髓干细胞悬浮液中,待细胞在TCP支架表面粘附,取出TCP支架,再将粘附有乳牙牙髓干细胞的TCP支架进行成骨诱导,得到所述3D打印牙根支架。
优选地,所述乳牙牙髓干细胞悬浮液的培养基为完全培养基,所述完全培养基含有α-MEM培养基、胎牛血清、青霉素和链霉素。
优选地,所述乳牙牙髓干细胞悬浮液的细胞浓度为4~6×105个/mL。
优选地,所述乳牙牙髓干细胞悬浮液的细胞浓度为5×105个/mL。
优选地,所述成骨诱导的时间为6~8天。
优选地,所述成骨诱导的时间为7天。
与现有技术相比,本发明的有益效果是:
本发明将生物陶瓷材料(β-磷酸三钙)、干细胞、3D打印技术的优势相互结合,将其联合应用于位点保存术上,3D打印的TCP支架具有良好的生物学特性,并且乳牙牙髓干细胞可作为负载其上的良好骨组织工程种子细胞;乳牙牙髓干细胞联合TCP支架材料可有效保存拔牙后的牙槽骨水平宽度及牙槽嵴颊侧骨板垂直高度,并促进牙槽窝内新骨的形成。
附图说明
图1为本发明实施例1的用于牙槽骨位点保存的3D打印牙根支架的宏观照片和SEM图。
图2为比格犬位点保存术试验中牙槽嵴高度变化的结果图。
图3为比格犬位点保存术试验中牙槽嵴宽度变化的结果图。
图4为比格犬位点保存术试验中牙槽嵴骨量变化的Micro-CT结果图。
具体实施方式
下面结合具体实施方式对本发明作进一步的说明。
实施例中的原料均可通过市售得到;
除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1
本实施例提供一种用于牙槽骨位点保存的3D打印牙根支架,该3D打印牙根支架为中空三维连通结构,主体材料为β-磷酸三钙,表面粘附有乳牙牙髓干细胞并进行成骨诱导。
本实施例的3D打印牙根支架的制备方法如下:
S1.通过锥形束投照计算机重组断层影像(corn bean computed tomography,CBCT)采集下颌骨数据,扫描层厚0.25mm,扫描完成后重建颌骨形态,保存标准dicom格式文件。将数据导入MIMICS 17.0,调整图像的阈值,优化使图像清晰完整。将处理完成的图像数据输出为STL格式,继续优化,仔细去除下颌P2-P4以外的组织,得到完整的前磨牙模型,采用切割功能分割牙根牙冠,将牙根数据生成新的STL文件。
S2.根据处理得到的三维模型,使用自动注浆技术打印TCP牙根支架。β-TCP粉末溶于蒸馏水中制成悬浮液(β-TCP粉末与水按体积比1∶1),随后加入C、羟丙基甲基纤维素和聚乙烯亚胺,进行分散、乳化和凝聚,形成打印的浆料,将牙根STL文件导入Robocad软件中设置打印参数,打印喷嘴直径200μm,打印速度12,相邻两层高度200μm,然后使用自动注浆打印机(3D Inks公司,美国)堆积成型。打印结束后,将支架放在室温下干燥24h。放入马弗炉中,2.5h匀速升至400℃后持续1h,去除有机物,最后,在4h内匀速升至1100℃,然后烧结3h,得到最终的支架,高温高压消毒后备用。
S3.收集新鲜拔除的健康滞留乳牙,选择牙根2/3以上的用拔髓针取牙髓组织,分离、剪碎,胶原酶及分散酶消化45min后离心,将组织块均匀置入T25培养瓶中,使用添加10%胎牛血清、1%青霉素/链霉素的α-MEM培养基后将T25倒置于37℃、5%CO2浓度的培养箱中培养,4h后翻面。每两天倒置显微镜下观察一次,细胞爬出后记为原代细胞(P0)。待细胞生长面积达培养皿底面积60%时进行传代,P3-P6用于后续步骤。
待细胞密度达70%时,吸除培养基,使用10%磷酸缓冲盐溶液(phosphate buffersaline,PBS)冲洗,胰酶消化1min,离心,倒去上清液,加入新鲜培养基,调整细胞浓度为5×105个/mL。将消毒灭菌后的支架浸泡在细胞悬液中,待细胞在支架表面粘附,取出支架,并在矿化培养基中成骨诱导一周,得到用于牙槽骨位点保存的3D打印牙根支架。使用时,植入拔完牙后的牙槽窝内。
形貌观察、结构测定
随机选取6个实施例1中打印好的TCP支架,Micro-CT测定孔径及孔隙率:在分辨率15μm、电压80kV、电流100μA、旋转扫描范围180°、铝0.5mm滤片扫描参数下获取样品数据;将扫描后数据导入机器自带的NRecon1.0软件进行重建,得到支架的断层图像并使用CTAn1.13对感兴趣区域(volume of interest,VOI)进行计算分析材料孔隙率。
Micro-CT扫描后将支架切割成小块,常规固定、干燥、喷金。使用SEM检测支架的显微结构,设置分辨率分别为1000、3000放大倍数,加速电压为15kV。
测试结果如图1所示,肉眼观察制备的3D-TCP牙根支架可见规则的宏观孔隙结构,相互连通。SEM观察显示TCP牙根的微观多孔隙结构规则,连通性良好,表面具有一定的粗糙度和微孔。均匀的孔隙结构,表面粗糙,有利于细胞粘附。SEM检测到支架侧壁及横断面孔径分别为50-100μm、350-400μm。Micro-CT测得TCP牙根的孔隙率为53.34±15.12%。
比格犬位点保存术试验:采用实施例1的3D打印牙根支架
1.试验过程
(1)实验动物
购于广东前沿科技有限公司,所有的动物均通过检疫合格。该动物实验得到广州市白云区龙归兴科动物养殖场伦理委员会的批准,生产许可证:SCXK(粤)2015-0037;使用许可证:SYXK(粤)2017-0171;动物实验伦理批准号:SYXK-2017070102
品系和等级:比格犬(beagle dog),普通级。
数量和性别:2只,雄性。
动物月龄:12~20月龄。
动物体重范围:12~16kg。
动物标识方法:供应商在耳内侧标识。
(2)实验动物分组
按照位点保存术方法,将比格犬拔牙后的24个位点随机分为3组,分别空白对照组、单纯TCP牙根组,TCP牙根+SHED组。在TCP牙根+SHED组,称重支架,将SHED按照106/g接种于TCP牙根上,矿化诱导一周;单纯TCP支架同时浸泡在矿化培养基中用于控制变量。
(3)动物模型的建立
动物购买后适应环境2周,饲养条件符合美国NIH和中国相关法律,动物的福利,包括住所、喂食、和环境,都进行了标准操作程序(SOP)。手术由动物中心的麻醉医师和护士的构成团队进行术中药物维持和护理。将比格犬单独饲养在不锈钢笼子内,25-27℃恒温,12h明暗交替。所有动物喂养专用比格犬饲料,提供充足的饮水。术后13周对动物进行处死,处死按照大动物安乐死的标准进行。实验犬术前禁食24h后行速眠新和戊巴比妥钠复合麻醉,方法是:
1)麻醉前15min,肌肉注射阿托品;
2)肌肉注射速眠新按体重每千克0.1毫升,进行基础麻醉;
3)基础麻醉后用2.5%戊巴比妥钠静脉注射按体重每千克0.3-0.4毫升。待比格犬呼吸平稳后进行手术,术中给予保暖、抗炎补液及密切监护:
常规消毒,铺巾,暴露术区(两侧下颂第二、三、四前磨牙),利多卡因局麻下选取微创拔牙挺配合下颌前磨牙钳分别拔除下颂第二、第三及第四前磨牙,使骨壁完整无破坏。所有拔牙窝均用刮匙搔刮干净,氯化钠注射液冲洗牙槽窝,按照(2)实验分组随机对牙槽窝进行处理,术后使用对位间断缝合法严密缝合,关闭拔牙创。
(4)术后处理
(4.1)术后检查
术后流食饲养2周,密切观察实验动物伤口愈合情况、精神状况、饮食及大小便情况。视情况给予抗炎、消肿止痛治疗。术后1周、6周、12周拍摄CBCT,观察愈合情况和骨结合情况。并于术后、5周、11周进行骨荧光素注射,追踪骨改建状态。术后13周给予比格犬安乐死并进行取材固定。
(4.2)CBCT拍摄及测量
将比格犬全麻后使用固定架固定进行CBCT扫描获取下颌骨数据,扫描层厚0.25mm,扫描完成后重建比格犬颌骨形态,保存标准dicom格式文件。
参照线的确定:根据比格犬下颌尖牙及第一磨牙牙尖进行平面定位,将平面拉至下颌骨下缘作为测量参照线。
实验位点的确定:通过术前CBCT,测好每个位点中部至第一前磨牙牙冠远中缘的距离,以此保证后续每次测量均在同一个位置。
实验位点牙长轴的确定:通过术前CBCT,连接牙尖及根尖的连线即为后续测量中的牙长轴。
实验位点颊舌侧垂直高度测量:在确定的位点截面图下分别从颊舌侧牙槽嵴顶向参照线作垂线,垂线长度在牙长轴上映射的距离作为颊侧骨板及舌侧骨板高度。
实验位点颊舌侧水平宽度的测量:分别测量舌侧牙槽嵴顶、舌侧牙槽嵴顶下2mm、舌侧牙槽嵴顶下4mm处的牙槽嵴宽度,测量线与牙长轴垂直。
(4.3)Micro-CT扫描
取备用标本,在分辨率25μm、电压80KV、电流100μA、旋转扫描范围180°、铝+铜滤片扫描参数下获取样品数据;将扫描后数据导入机器自带的NRecon 1.0软件进行重建,得到支架的断层图像。
Micro-CT重建:选取位点图像,使用CTvol 2.0软件重建位点处三维图像。
2.试验结果
(1)临床检查
所有实验动物均健康存活至处死。术后7d基本恢复正常饮食和活动量,伤口愈合良好。术后2周两组支架发生暴露,取出支架压迫止血,其余组均良好。
比格犬位点保存术后13周处死,所有位点的粘膜均愈合良好、色泽正常,未发现红肿、感染、坏疽及其他异常症状,材料存留率88.9%。
(2)CBCT结果
(2.1)垂直高度变化
颊侧骨板:如图2A所示,CBCT结果显示第6周时,TCP+SHED组颊侧骨板高度减少为0.04±0.27mm、单纯TCP组减少为0.33±0.17mm、空白对照组减少为1.47±0.51mm,方差分析显示TCP+SHED组与单纯TCP、空白对照组间比较均具有显著统计学差异(P<0.001);在第12周时,TCP+SHED组颊侧骨板高度减少为0.11±0.29mm、单纯TCP组减少为1.03±0.37mm、空白对照组减少为2.41±0.54mm,方差分析显示三组间均具有显著统计学差异(P<0.001)。
舌侧骨板:如图2B所示,CBCT结果显示第6周时,TCP+SHED组颊侧骨板高度减少为0.18±0.20mm、单纯TCP组减少为0.26±0.17mm、空白对照组减少为0.34±0.39mm,方差分析显示三组间无明显统计学差异(P>0.05);在第12周时,TCP+SHED组颊侧骨板高度减少为0.22±0.24mm、单纯TCP组减少为0.70±0.57mm、空白对照组减少为0.54±0.39mm,方差分析显示三组间无明显统计学差异(P>0.05)。
(2.2)水平宽度变化
舌侧牙槽嵴顶:如图3A所示,CBCT结果显示第6周时,TCP+SHED组舌侧牙槽嵴顶减少为0.01±0.15mm、单纯TCP组减少为0.53±0.22mm、空白对照组减少为1.36±0.63mm,方差分析显示TCP+SHED组与单纯TCP、空白对照组间比较均具有显著统计学差异(P<0.05);在第12周时,TCP+SHED组颊侧骨板高度减少为0.09±0.42mm、单纯TCP组减少为1.26±0.18mm、空白对照组减少为1.67±0.53mm,方差分析显示TCP+SHED组与单纯TCP组、空白对照组间均具有显著统计学差异(P<0.001),单纯TCP组与空白对照组间无统计学差异(P>0.05)。
舌侧牙槽嵴顶下2mm:如图3B所示,CBCT结果显示第6周时,TCP+SHED组颊侧骨板高度减少为0.07±0.21mm、单纯TCP组减少为0.36±0.35mm、空白对照组减少为0.38±0.36mm,方差分析显示三组间无明显统计学差异(P>0.05);在第12周时,TCP+SHED组颊侧骨板高度减少为0.19±0.35mm、单纯TCP组减少为0.60±0.44mm、空白对照组减少为0.71±0.45mm,方差分析显示SHED+TCP组与空白对照组间有显著统计学差异(P<0.05),单纯TCP组与SHED+TCP组、空白对照组间无明显统计学差异(P>0.05)。
舌侧牙槽嵴顶下4mm:如图3C所示,CBCT结果显示第6周时,TCP+SHED组颊侧骨板高度减少为0.01±0.22mm、单纯TCP组减少为0.06±0.20mm、空白对照组减少为0.15±0.24mm,方差分析显示三组间无明显统计学差异(P>0.05);在第12周时,TCP+SHED组颊侧骨板高度减少为0.05±0.22mm、单纯TCP组减少为0.18±0.22mm、空白对照组减少为0.17±0.22mm,方差分析显示三组间无明显统计学差异(P>0.05)。
(3)Micro-CT结果
Micro-CT三维重建的结果如图4所示,在13周后的Micro-CT重建结果中可发现未经处理的空白组(图4A)位点颊侧骨板出现明显的吸收,由颊侧牙槽嵴顶开始向下向内凹陷;单纯TCP牙根组(图4B)则由于材料的支撑在整体轮廓上变化不大,但颊侧骨壁出现吸收;而SHED牙根组(图4C)不仅在轮廓上基本维持了原来位点的外形,颊侧骨板也得到了有效的保存,且在一些组中发现在支架顶部有新骨包绕。
显然,本发明的上述实施例仅仅是为清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一种用于牙槽骨位点保存的3D打印牙根支架,其特征在于,所述3D打印牙根支架为中空三维连通结构,主体材料为β-磷酸三钙,表面粘附有乳牙牙髓干细胞并进行成骨诱导。
2.根据权利要求1所述的3D打印牙根支架,其特征在于,所述3D打印牙根支架上乳牙牙髓干细胞的粘附量为105~7个/g。
3.根据权利要求2所述的3D打印牙根支架,其特征在于,所述3D打印牙根支架上乳牙牙髓干细胞的粘附量为106个/g。
4.根据权利要求1所述的3D打印牙根支架,其特征在于,所述3D打印牙根支架的侧壁孔径为50~100μm,横断面孔径为350~400μm,孔隙率为50%~60%。
5.权利要求1~4任一项所述3D打印牙根支架的制备方法,其特征在于,包括如下步骤:
S1.根据待保存位点的形状建立三维模型;
S2.配制主要含有β-磷酸三钙的打印浆料,通过3D打印机根据步骤S1的三维模型打印、烧结得到TCP支架;
S3.将步骤S2的TCP支架浸泡在乳牙牙髓干细胞悬浮液中,待细胞在TCP支架表面粘附,取出TCP支架,再将粘附有乳牙牙髓干细胞的TCP支架进行成骨诱导,得到所述3D打印牙根支架。
6.根据权利要求5所述的制备方法,其特征在于,所述乳牙牙髓干细胞悬浮液的培养基为完全培养基,所述完全培养基含有α-MEM培养基、胎牛血清、青霉素和链霉素。
7.根据权利要求5所述的制备方法,其特征在于,所述乳牙牙髓干细胞悬浮液的细胞浓度为4~6×105个/mL。
8.根据权利要求5所述的制备方法,其特征在于,所述乳牙牙髓干细胞悬浮液的细胞浓度为5×105个/mL。
9.根据权利要求5所述的制备方法,其特征在于,所述成骨诱导的时间为6~8天。
10.根据权利要求7所述的制备方法,其特征在于,所述成骨诱导的时间为7天。
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