生物陶瓷材料负载冻干BMP-2对牙槽突裂骨改建的影响

目的： 负载冻干BMP-2到一新型无机生物陶瓷类支架材料OsteoBoneTM，验证其对组织工程骨修复牙槽突裂成骨作用的影响。方法： 通过冻干法并添加海藻糖作为冻干保护剂，将BMP-2与OsteoBoneTM支架材料相结合；之后置于PBS溶液中并收集缓释液，BCA法检测随时间变化的蛋白浓度，ALP检测成骨诱导活性。选取18只8周龄雄性SD大鼠，随机分为3组，每组6只，在大鼠左右侧牙槽骨上人为制作4 mm×3 mm×3 mm牙槽突裂，将单纯材料（A组）、BMSC-材料复合物（B组）与构建出的BMP-2-材料复合物（C组），分别植入大鼠牙槽骨骨缺损区。分别于术后3周与6周取材，H-E染色进行组织学观察，比较3组之间的成骨差异，并进行micro-CT影像学分析。采用SPSS19.0软件包对实验数据进行统计学分析。结果： 通过冻干法并添加海藻糖作为冻干保护剂，将BMP-2与OsteoBoneTM支架材料结合后，BMP-2-材料复合物具备持续的随时间渐增的缓释BMP-2的能力，且表现出明显的ALP活性。体内实验术后3周，A、B、C组H-E染色观察可见较稀疏编织骨，3组间无显著差异；micro-CT分析BV/TV和骨小梁厚度，3组间无显著差异。术后6周，组织学观察B组与C组新骨形成趋势比A组更明显；micro-CT分析BV/TV和骨小梁厚度，B、C组显著大于A组，B组与C组间无显著差异。结论： 负载冻干BMP-2到OsteoBoneTM支架材料能显著提高其成骨能力，达到与间充质干细胞相近的效果。     

骨髓间充质干细胞联合纤维蛋白胶修复大鼠牙槽骨缺损的研究

目的 评价骨髓间充质干细胞(BMSCs)和纤维蛋白胶(FG)作为种子细胞和支架材料修复大鼠牙槽骨缺损的效果.方法 30只SD大鼠随机分成A、B组,建立SD大鼠上颌牙槽骨缺损模型,联合植入BMSCs和FG复合物,A组对照侧单纯植入FG,B组对照侧为空白对照.利用上颌骨实验区切片苏木精-伊红染色及Micro-CT扫描观察成...     

负压与BMSCs膜片促进细胞定植和体内成骨的探讨

目的:研究经过负压处理的TCP支架材料与细胞复合后的体内成骨能力。方法:将TCP支架材料负压处理,和骨髓基质细胞(BMSCs)细胞共培养一段时间后,成骨诱导2周,电镜下观察支架材料和细胞复合情况。再将该复合物与BMSCS细胞膜片复合,植入到裸鼠皮下,8周后作组织切片,HE染色和Masson染色,观察其体内成骨能力。结果:负压(有细胞)+膜片组异位移植8周后,内部可见大量胶原纤维和成骨纤维,在团块样组织内部可见大量成骨细胞,并有血管形成。对照组仅有个别细胞密集区,胶原纤维和成骨纤维较少。空白组未见类似情况。结论:经过负压+骨髓基质细胞膜片处理的TCP支架材料,在体内有成骨潜能。     

脐带间充质干细胞复合β-磷酸三钙生物陶瓷体内外成骨能力的初步研究

目的:观察人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)和支架材料β-磷酸三钙(β-TCP)生物陶瓷复合的体内外成骨情况。方法:在体外构建hUCMSCs和β-TCP的复合物,将细胞以3×105/mL的浓度接种到支架材料上进行复合体构建,并进行电镜观察、特异性免疫荧光染色、MTT、ALP检测。将复合物植入裸鼠体内,进行成骨能力研究。实验分为3组,即植入单纯β-TCP支架组、植入体外成骨诱导2周的hUCMSCs和β-TCP复合物组、单纯植入体外成骨诱导2周的hUCMSCs组。植入后2个月取出标本,进行大体观察、X线片观察和组织学观察。采用SPSS16.0软件包对实验数据进行重复测量随机区组设计的方差分析。结果:hUCMSCs植入支架4h后,即可见细胞在支架上附着,1周后可见细胞在支架中大量增殖,β-TCP具有一定的骨诱导性。复合物植入裸鼠内2个月,hUCMSCs和β-TCP复合组X线密度最高。HE染色显示,2个月后,hUCMSCs和β-TCP复合组见不规则新骨和血管形成,单纯β-TCP组未见明显新骨和血管形成。Masson染色显示,2个月后,hUCMSCs和β-TCP复合组见大量胶原形成,单纯β-TCP组未见明显胶原形成。VG染色显示,2个月后,hUCMSCs和β-TCP复合组孔隙中有大量类骨质及少量骨陷窝形成,单纯β-TCP组未见类骨质形成。结论:hUCMSCs和β-TCP具有良好的生物相容性,两者构建的复合物在植入裸鼠体内2个月时可见新骨及血管化形成。     

羟基磷灰石与骨髓基质细胞自体皮下成骨实验

目的 :观察rhBMP2 /TGF - β对羟基磷灰石与骨髓基质细胞在自体皮下成骨能力的影响。 方法 :应用矿化条件培养液体外培养成年新西兰兔的骨髓基质细胞 ,收集细胞并以 5× 10 6/mL浓度分成 4组 ,A组加入rhBMP2(10 μg) /TGF - β(2 0ng) ,B组加入rhBMP2 (10 μg) ,C组不加生长因子作为对照 ,然后将细胞接种在羟基磷灰石上 ,体外培养 3d ,D组细胞不接种材料上 ,继续体外培养。组织化学方法检测检测磷酸酶 ,扫描电镜观察细胞在材料上的附着情况。将复合物埋植于新西兰兔自体皮下。 4周后常规HE染色、免疫组织化学染色并进行图像分析观察Ⅰ型胶原、骨形成蛋白合成情况。结果 :A、B组的碱性磷酸酶平均OD值显著高于C组 (P <0 .0 1)。细胞接种在材料 3d后 ,各组材料表面均见大量多边形的细胞附着。复合物植入皮下 4周时HE染色见各组均有条索状骨基质形成 ,A、B组Ⅰ型胶原平均灰度值明显低于C组 (P <0 .0 1=,各组骨形成蛋白的表达无明显差异 (P >0 .0 5 )。结论 :羟基磷灰石与骨髓基质细胞在自体皮下能够形成骨组织 ,rhBMP2 /TGF - β较rhBMP2 有更强的促进骨形成作用     

异种脱钙骨兔体内成骨能力的实验研究

目的制备猪脱钙骨,将兔骨髓间质干细胞与猪脱钙骨支架形成细胞-生物材料复合物,回植入兔皮下观察该材料体内成骨能力。方法梯度离心法分离兔骨髓基质干细胞,向成骨细胞方向诱导培养。将猪颅骨用氯仿丙酮及过氧化氢处理后冻干保存,钴60照射消毒待用。制备细胞-生物材料复合物后植入兔皮下,分别在4w,8w,12w取材,同期大体观察及组织学检测,切片HE染色。结果倒置相差显微镜下见细胞在材料孔隙间大量生长增殖、交叠覆盖;扫描电镜观察细胞贴附于材料表面并向孔隙内生长。组织学检测4w实验组见多数骨小梁表面有细胞被覆,有较多小血管形成。8w、12w实验组见新生骨小梁有新生骨组织形成,见成骨细胞和破骨细胞并存。结论兔骨髓间质干细胞与猪脱钙骨有良好的生物相容性,在该支架材料上细胞增殖分化并具有一定的体内成骨能力。     

载辛伐他汀纳米羟基磷灰石支架材料对兔下颌骨缺损修复的影响

目的:采用骨髓间充质干细胞(BMSCs)复合辛伐他汀支架材料修复兔下颌骨局部缺损,观察辛伐他汀对BMSCs复合支架材料修复兔骨缺损的影响。方法:24只新西兰大白兔随机分为2组,每组12只,制备下颌骨缺损模型。A组为实验组,在缺损区植入复合BMSCs的载辛伐他汀纳米羟基磷灰石支架材料;B组为对照组,植入BMSCs复合羟基磷灰石材料。分别于术后2、4、8、12周处死各组动物,行影像学分析、HE染色、扫描电镜观察等检查。结果:影像学检查及组织学染色结果显示,A组骨缺损处愈合程度、成骨速度及骨质量明显优于B组。扫描电镜显示,A组复合材料与组织相容性好,材料吸收优于B组。统计各组各期牙CT分析数据的骨密度值,结果表明A组的骨密度值明显高于B组(P<0.05)。结论:实验表明经BMSCs复合的辛伐他汀支架材料具有明显的促进成骨能力,可加速骨缺损的修复效果,提高修复质量。     

骨钙素与低强度激光对正畸牙移动速度的影响研究

目的 探讨骨钙素和低强度激光照射作用对正畸牙移动的影响.方法 取96只SD大鼠,分为A、B、C、D四个组,每组24只.A组:放置矫治装置后,每天在大鼠加力磨牙腭侧注入1 mg大鼠纯化骨钙素.B组:在放置矫治装置后,每天用弱激光照射被移动的磨牙.C组:在放置矫治装置的当天,每天在大鼠加力磨牙腭侧注入1 mg骨钙素,同时用弱激光照射.D组(对照组):只放置矫治装置给大鼠磨牙加力,不采取其他措施.分别测量各组大鼠加力后1、3、5、7、10、14 d牙齿的移动距离,并进行苏木精-伊红染色观察.结果 C组大鼠牙移动的距离明显大于其他3个组(P＜0.05);与D组比较,C组压力侧破骨现象明显,张力侧成骨活跃.结论 骨钙素与激光共同作用有效地加速了实验性大鼠的正畸牙移动.     

邻牙移入对组织工程骨修复的牙槽突裂区骨改建的影响

目的研究利用3D打印的支架材料制作组织工程骨并植入大鼠牙槽突裂区,邻牙受正畸力向植骨区移动对植入骨改建的生物学影响。方法选取12只8周龄SD大鼠,制备双侧上颌牙槽突裂标准动物模型。利用3D打印的组织工程支架材料与大鼠骨髓间充质干细胞（BMSCs）体外共培养后,作为植入材料,植入动物体内。术后8周处死3只大鼠取材, H-E染色评价成骨,并行单侧植骨区邻牙加力移入,非加力侧为对照组。在加力1、3、7 d后分别处死3只大鼠取材,采用实时荧光定量PCR技术（RT-PCR）检测成骨相关基因Runx2、OCN和VEGF的表达,应用SPSS19.0软件包对实验组和对照组的基因表达样本均数进行t检验。结果H-E染色显示,手术区骨组织成熟,成骨良好。RT-PCR显示,Runx2呈现早期到中期增高,而到晚期再降低的趋势;OCN的表达在早期到中期无显著变化,到晚期升高明显;VEGF表达在加力早期到中期再到晚期呈现逐渐降低的趋势;而早、中、晚期实验组的各项成骨相关基因表达均显著高于对照组（P<0.05）。结论3D打印支架材料构建的组织工程骨可以很好地替代自体骨移植,以达到良好的成骨效果。正畸手段使邻牙移入植骨区,可以有效促进术区成骨,并且诱导成骨细胞及破骨细胞进行骨改建。     

骨髓基质成骨细胞-松质骨基质复合人工骨皮下移植成骨作用的实验研究

目的：观察骨髓基质成骨细胞－松质骨基质复合人工骨皮下移植的成骨作用，探索骨髓基质成骨细胞和松质骨基质分别作为骨组织工程种子细胞和支架材料的可行性。方法：成年新西兰兔骨髓细胞体外培养，诱导后，经混匀，接种，固化，形成骨髓基质成髓细胞－藻酸盐－松质骨基质复合人工骨，并植回取材兔背部皮下，对照组分别植入藻酸盐－松质骨基质复合物和松质骨基质，植入4，8周取材，行X线摄片，组织学检查和组织学定量分析，观察骨形成情况，结果：骨髓基质成骨细胞－藻酸盐－松质骨基质复合人工骨皮下成骨效果明显优于藻酸盐－松质骨基质复合物组和松质骨基质组，复合人工骨标本兼有膜内成骨和软成骨，以膜内成骨为主，对照组仅见不得软骨成骨，结论：采用组织工程方法形成的复合人工骨皮下成骨作用明显，骨髓基质成细胞和松质骨基质可以分别作为种子细胞和支架材料而用于组织工程骨的构建。     

Evaluation of the Apical Complex and the Coronal Pulp as a Stem Cell Source for Dentin-pulp Regeneration

IntroductionThis study compared the stemness and differentiation potential of stem cells derived from the apical complex (apical complex cells [ACCs]) and coronal pulp (dental pulp stem cells [DPSCs]) of human immature permanent teeth with the aim of determining a more suitable source of stem cells for regeneration of the dentin-pulp complex.MethodsACC and DPSC cultures were established from 13 human immature permanent teeth using the outgrowth method. The proliferation capacity and colony-forming ability of ACCs and DPSCs were evaluated. ACCs and DPSCs were analyzed for mesenchymal stem cell markers using flow cytometry. The adipogenic and osteogenic differentiation potential of ACCs and DPSCs were evaluated using the quantitative real-time polymerase chain reaction and histochemical staining. ACCs and DPSCs were transplanted subcutaneously in immunocompromised mice using macroporous biphasic calcium phosphate as a carrier. The histomorphologic characteristics of the newly formed tissues were verified using hematoxylin-eosin staining and immunohistochemical staining. Quantitative alkaline phosphatase analysis and quantitative real-time polymerase chain reaction using BSP, DSPP, POSTN, and Col XII were performed.ResultsACCs and DPSCs showed similar cell proliferation potential and colony-forming ability. The percentage of mesenchymal stem cell markers was similar between ACCs and DPSCs. In the in vitro study, ACCs and DPSCs showed adipogenic and osteogenic differentiation potential. In the in vivo study, ACCs and DPSCs formed amorphous hard tissue using macroporous biphasic calcium phosphate particles. The quantity and histomorphologic characteristics of the amorphous hard tissue were similar in the ACC and DPSC groups. Formation of periodontal ligament–like tissue, positive to Col XII, was observed in ACC transplants, which was absent in DPSC transplants.ConclusionsACCs and DPSCs showed similar stemness, proliferation rate, and hard tissue–forming capacity. The notable difference was the periodontal ligament–like fiber-forming capacity of ACCs, which indicates the presence of various lineages of stem cells in the apical complex compared with the coronal pulp. Regarding regeneration of the dentin-pulp complex, the coronal pulp can be a suitable source of stem cells considering its homogenous lineages of cells and favorable osteo/odontogenic differentiation potential.     

载辛伐他汀PLGA/CPC复合骨髓基质干细胞构建组织工程骨的实验研究

目的：探讨载辛伐他汀PLGA/CPC复合骨髓基质干细胞(bone marrow stromal stem cells)构建组织工程骨的可行性,并筛选辛伐他汀的有效载药量。方法：采用溶剂浇铸—粒子沥滤技术结合相分离法,制备不同浓度(辛伐他汀质量分别为0.1、0.5、1 mg)的载辛伐他汀PLGA/CPC复合支架材料,扫描电镜观察孔隙率,绘制药物释放曲线;茜素红染色、I型胶原染色观察成骨诱导液和辛伐他汀对骨髓基质干细胞向成骨分化的作用;将第3代BMSCs经dil染色后,接种于不同浓度的复合支架材料上,扫描电镜、激光共聚焦显微镜观察细胞在支架上的黏附情况,CCK-8和碱性磷酸酶(ALP)检测对其增殖和分化作用;采用SPSS 18.0软件包对数据进行统计学分析。结果：支架材料孔隙率达90%以上,孔径平均200~300 μm,载药组药物持续缓慢释放,未见药物突释现象;经辛伐他汀和成骨诱导组I型胶原表达阳性,茜素红染色可见明显钙结节;4组支架材料与细胞黏附性较好,0.5 mg组细胞生长状态最佳。CCK-8和ALP检测0.5 mg组能够明显促进细胞的增殖和分化。结论：辛伐他汀和成骨诱导液联合利用,能够更有效地促进BMSCs向成骨分化;载辛伐他汀PLGA/CPC复合支架材料是理想的组织工程支架材料;载0.5 mg辛伐他汀PLGA/CPC支架材料能够有效促进BMSCs的增殖和分化。     

Enhancing cell seeding and osteogenesis of MSCs on 3D printed scaffolds through injectable BMP2 immobilized ECM-Mimetic gel

ObjectiveDesign of bioactive scaffolds with osteogenic capacity is a central challenge in cell-based patient-specific bone tissue engineering. Efficient and spatially uniform seeding of (stem) cells onto such constructs is vital to attain functional tissues. Herein we developed heparin functionalized collagen gels supported by 3D printed bioceramic scaffolds, as bone extracellular matrix (ECM)-mimetic matrices. These matrices were designed to enhance cell seeding efficiency of mesenchymal stem cells (MSCs) as well as improve their osteogenic differentiation through immobilized bone morphogenic protein 2 (BMP2) to be used for personalized bone regeneration.MethodsA 3D gel based on heparin-conjugated collagen matrix capable of immobilizing recombinant human bone morphogenic protein 2 (BMP2) was synthesized. Isolated dental pulp Mesenchymal stem cells (MSCs) were then encapsulated into the bone ECM microenvironment to efficiently and uniformly seed a bioactive ceramic-based scaffold fabricated using additive manufacturing technique. The designed 3D cell-laden constructs were comprehensively investigated trough in vitro assays and in vivo study.ResultsIn-depth rheological characterizations of heparin-conjugated collagen gel revealed that elasticity of the matrix is significantly improved compared with freely incorporated heparin. Investigation of the MSCs laden collagen-heparin hydrogels revealed their capability to provide spatiotemporal bioavailability of BMP2 while suppressing the matrix contraction over time. The in vivo histology and real-time polymerase chain reaction (qPCR) analysis showed that the designed construct supported the osteogenic differentiation of MSCs and induced the ectopic bone formation in rat model.SignificanceThe presented hybrid constructs combine bone ECM chemical cues with mechanical function providing an ideal 3D microenvironment for patient-specific bone tissue engineering and cell therapy applications. The implemented methodology in design of ECM-mimetic 3D matrix capable of immobilizing BMP2 to improve seeding efficiency of customized scaffolds can be exploited for other bioactive molecules.     

PEGS/β-TCP屏障膜在促进大鼠颅骨缺损骨组织再生中的作用

目的 通过体内和体外实验,探讨PEGS/β-TCP复合膜诱导骨组织再生的效果。方法 将PEGS/β-TCP复合膜与大鼠骨髓间充质干细胞（rBMSCs）共培养,观察rBMSCs细胞黏附、增殖及成骨分化情况。建立大鼠颅骨缺损动物模型,将PEGS/β-TCP复合膜植入缺损特定区域,通过Micro-CT观察骨缺损区域新骨形成情况;再通过脱钙组织H-E染色,观察炎性浸润程度和新骨形成的情况。采用SPSS 22.0软件包对数据进行统计学分析。结果 rBMSCs可良好黏附和广泛分布于PEGS/β-TCP复合膜上。rBMSCs接种1、4和7 d后进行的dsDNA定量分析结果显示,从第1天到第7天,细胞DNA含量逐渐增加;培养7 d后,实验组细胞DNA含量显著高于对照组（P<0.001）。对接种于PEGS/β-TCP复合膜上的rBMSCs进行体外成骨诱导,其碱性磷酸酶（ALP）染色浓度显著增高。ALP活性半定量检测显示,实验组较对照组细胞ALP活性增加更为显著（P<0.01）。成骨诱导21 d后,实验组与对照组茜素红矿化染色结果与ALP染色结果一致。动物模型Micro-CT检测及组织H-E染色均显示,实验组各检测时间点的新骨形成量显著高于对照组,且各组炎症反应均轻微可控。结论 PEGS/β-TCP复合膜具有理想的细胞相容性和骨引导再生效果。     

The Effect of Varying Occlusal Loading Conditions on Stress Distribution in Roots of Sound and Instrumented Molar Teeth: A Finite Element Analysis

IntroductionThis study aimed to investigate whether the direction of force applied to the occlusal surface influenced the pattern of tensile stresses in roots of sound and root canal–prepared mandibular molar teeth. The effect of obturation forces on the development of apical stress was also investigated. To this end, models were constructed using micro–computed tomographic imaging and investigated using finite element analysis.MethodsMicro–computed tomographic data established boundaries of internal and external model surfaces to allow finite element analysis. Individually segmented components were modeled based on mechanical properties in precedent literature. The following conditions were considered: axial force directed over the mesial marginal ridge, a mesial or a distal tipping force, a combination of both a torquing force and axial loading, and hydrostatic pressure. The maximum principal stresses were determined.ResultsThe highest root stress occurred in the cervical third of root surfaces (ie, not apically) under all loading conditions. Importantly, mesial tipping forces resulted in tension on distal roots, whereas distal tipping resulted in tension in the mesial roots. Intracanal pressures produced tensile stress on the internal root canal walls in the cervical third of the root. Stresses were calculated to be less than the fatigue tensile strength of dentin.ConclusionsStatic loading, under the conditions modeled, does not result in stress concentration at the root apices that would cause root fracture under normal masticatory loads. Stress patterns developing from mesial and distal tipping forces help to explain the appearance of vertical root fractures reported in sound nonrestored molar teeth.     

Chondrogenic potential of stem cells from human exfoliated deciduous teeth in vitro and in vivo

Objective. The aim of this study was to investigate the chondrogenic potential of stem cells from human exfoliated teeth (SHED). Materials and methods. SHED cultures were isolated from human exfoliated deciduous teeth. Colony-forming capacity, odonto/osteogenic and adipogenic potential were measured. SHED were cultured for 2 weeks in chondrogenic differentiation medium containing dexamethasone, insulin, ascorbate phosphate, TGF-β3 and bFGF. Toluidine blue staining and safranin O staining were used for chondrogenesis analysis. The related markers, type II collagen and aggrecan, were also investigated using immunohistochemistry. SHED were seeded onto the β-TCP scaffolds and transplanted into the subcutaneous space on the back of nude mice. The transplants were recovered at 2, 4 and 8 weeks post-transplantation for analysis. Results. SHED showed colony-forming capacity, odonto/osteogenic and adipogenic differentiation capacity. Chondrogenic differentiation was confirmed by toluidine blue staining, safranin O staining, type II collagen and aggrecan immunostaining. After in vivo transplantation, SHED recombined with β-TCP scaffolds were able to generate new cartilage-like tissues. Conclusions. The ?ndings demonstrate the chondrogenic differentiation capacity of SHED both in vitro and in vivo models, suggesting the potential of SHED in cartilage tissue engineering.