3D打印聚乙烯醇/纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的性能比较

目的　采用3D打印技术制备3D打印聚乙烯醇 /纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架,并对其进行表征。方法　采用3D打印技术制作聚乙烯醇/纳米羟基磷灰石支架以及丝素蛋白/聚乙烯醇/纳米羟基磷灰石复合支架。进行孔隙率、扫描电镜、压缩力学性能及细胞毒性检测。 结果　①扫描电镜观察：丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架结构规则,网状结构清晰,交通支连续,层层之间搭接良好,支架空隙均一。相同倍数下,聚乙烯醇/纳米羟基磷灰石支架网状结构连续性较差。②压缩力学性能：相同应力情况下（10 MPa）,3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的应变大于3D打印聚乙烯醇/纳米羟基磷灰石支架。③孔隙率：3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的孔隙率大于3D打印聚乙烯醇/纳米羟基磷灰石支架。④细胞毒性检测：不同时间点两组支架的细胞增殖率无明显差别。结论　结果表明：3D打印聚乙烯醇 /纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架具有良好的理化性能和细胞相容性。     

Dickkopf-1 may regulate bone coupling by attenuating wnt/β-catenin signaling in chronic apical periodontitis

ObjectiveAlveolar bone loss is a common outcome of chronic apical periodontitis. In this study, we investigated the involvement of the Dickkopf-1-Wnt/β-catenin signaling pathway in the attenuation of osteogenic differentiation induced by Escherichia coli lipopolysaccharide, and we evaluated the use of Dickkopf-1 inhibitor and Dickkopf-1 recombinant protein to reverse bone loss in different phases of osteogenic differentiation.MethodsMC3T3-E1 cells grown in osteogenic medium were treated with Escherichia coli lipopolysaccharide for 24 h during osteogenic induction on days 0, 1, 7, 14 and 21. Dickkopf-1 siRNA was added on days 0 and 1, and Dickkopf-1 recombinant was added on days 7, 14, and 21. Quantitative real-time PCR, Western blotting and alkaline phosphatase activity assays were performed to measure osteogenic marker expression and Wnt/β-catenin signaling. A rat apical periodontitis model was used to further evaluate the function of Dickkopf-1 in relation to bone loss.ResultsMC3T3-E1 cells treated with Escherichia coli lipopolysaccharide showed decreased mRNA expression of osteogenic markers. Wnt/β-catenin signaling was also inhibited, and Dickkopf-1 showed corresponding variations as quantified by Western blotting. Using Dickkopf-1 inhibitor or Dickkopf-1 recombinant protein at different phases of osteogenic differentiation in vitro partially reversed the decrease in osteogenic marker expression. The rat apical periodontitis model indicated that the Dickkopf-1 inhibitor could restore bone loss in the periapical area in vivo.ConclusionsDickkopf-1 may play a key regulatory role in determining the outcome for bone in inflammatory environments, and modulating the Wnt/β-catenin signaling pathway via Dickkopf-1 inhibitor or recombinant protein may provide a potential therapeutic option to prevent bone destruction in endodontic disease.     

釉基质蛋白诱导MC3T3-E1细胞成骨分化过程中微小核糖核酸的表达

目的 探讨微小核糖核酸(micro ribonucleicacid,miRNA)是否参与到釉基质蛋白(EnamelMatrix Derivative,EMD)诱导的小鼠前成骨细胞系MC3T3-E1细胞成骨分化的过程,并对发生显著变化的miRNA进行分析.方法 将MC3T3-E1用含EMD(刺激组)/不含EMD(对照组)的培养液培养0天、7天和14天,检测碱性磷酸酶(alkaline phosphatase,ALP)活性,实时聚合酶链式反应(RT-PCR)技术分析成骨分化标记物ALP、骨涎蛋白(bone sialoprotein,BSP)和骨钙素(osteocalcin,OC)的信使RNA(mRNA)水平的表达变化.利用基因芯片技术分析miRNA的相对表达.结果 0天、7天、14天的ALP染色结果显示随着培养时间的增加,两组ALP活性均逐渐增强,EMD刺激组ALP活性增强较对照组更为明显.RT-PCR结果表明,与对照组相比,ALP、BSP、OC的mRNA表达量均显著增加,ALP与OC均于14天时表达量达到峰值,BSP则于7天时处于峰值表达,EMD刺激组MC3T3-E1成骨活性更强;各期11个miRNA表达上调,28个miRNA表达下调,其中miR-335-5p,miR-503已被证实可参与促进骨形成,而miR-30家族(miR-30a,-30b,-30c和-30d)则被证实参与抑制成骨.结论 miRNA参与EMD诱导的MC3T3-E1细胞的成骨分化过程,这一发现可以为了解EMD促进成骨分化的机理及临床应用提供指导.     

骨髓间充质干细胞与支架材料nHA/PA66复合培养的生物活性研究

目的：研究骨髓间充质干细胞与支架材料-纳米羟基磷灰石/聚酰胺66（nHA/PA66）复合培养时生物学特性。方法：体外培养兔骨髓间充质干细胞（MSCs）,并分为A、B、C、D四组。A组为对照;B组和D组进行骨向诱导;同时,C组和D组细胞与nHA/PA66复合培养。应用MTT实验、碱性磷酸酶（ALP）染色和生物化学分析、扫描电镜,分别对各组细胞的增殖速度、ALP活性及细胞在支架上的黏附情况进行检测。结果：MTT结果显示,A组细胞增殖曲线与C组相似,而B组和D组相似;A组和C组细胞增殖略高于B组和D组。结果表明,骨向诱导可使细胞增殖轻微下降,而与支架材料复合培养对MSCs增殖无影响。ALP染色及ALP活性生化分析进一步表明,MSCs与支架材料复合培养对其骨向分化能力无影响。SEM观察则表明,C组细胞和D组细胞与支架材料均粘附良好。结论：nHA/PA66适于MSCs的黏附、增殖及骨向分化,是一种极具价值的骨组织工程支架材料。     

Sodium hyaluronate accelerates the healing process in tooth sockets of rats

Objective

In this study we evaluated the effects of sodium hyaluronate (HY) in the healing process of tooth sockets of rats.

Design

Immediately after the extraction of the upper first molars of male Holtzman rats, right sockets were treated with 1% HY gel (∼0.1 ml), while left sockets were used as control (blood clot). The animals were sacrificed at 2, 7, and 21 days after tooth extraction and upper maxillaries processed for histological and morphometric analysis of the apical and medium thirds of the sockets. Carbopol, an inert gel, was used to evaluate the mechanical effect of gel injection into sockets. Expression of bone morphogenetic protein-2 (BMP-2) and osteopontin (OPN) was determined by immunohistochemistry at 1, 2, 3, 4, 5, and 7 days after tooth extraction.

Results

Histological analysis showed that HY treatment induced earlier trabecular bone deposition resulting in a bone matrix more organized at 7 and 21 days after tooth extraction. Also, HY elicited significant increase in the amount of bone trabeculaes at 7 and 21 days after tooth extraction (percentage of trabecular bone area at 7 days: 13.21 ± 4.66% vs. 2.58 ± 1.36% in the apical third of control sockets) and in the vessels counting at 7 days. Conversely, the number of cell nuclei was decreased in HY-treated sockets. Additionally, expression of BMP-2 and OPN was enhanced in HY-treated sockets compared with control sockets.

Conclusions

These findings suggest that HY accelerates the healing process in tooth sockets of rats stimulating the expression of osteogenic proteins.     

Three-dimensional culture of rat BMMSCs in a porous chitosan-gelatin scaffold: A promising association for bone tissue engineering in oral reconstruction

Objective

This study investigated the in vitro effects of a chitosan-gelatin scaffold on growth and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMMSCs) in three-dimensional (3D) cultures and evaluated the biomaterial biocompatibility and degradability after its grafting into tooth sockets of rats.

Design

A porous chitosan-gelatin scaffold cross-linked by glutaraldehyde was synthesised and characterised by light (LM), scanning electronic microscopy (SEM), energy dispersion spectroscopy (EDS) and X-ray diffraction (XRD). Rat BMMSCs were isolated, expanded and seeded onto scaffold using Dulbecco's Modified Eagle's Medium (DMEM) with or without an osteogenic supplement. Cell viability by MTT assay, alkaline phosphatase (ALP) activity and morphological LM and SEM analysis were performed after 1, 3, 8 and 14 days in culture. Free-cell scaffolds were implanted into tooth sockets of Lewis rats after upper first molars extraction. Fifteen male recipient rats were sacrificed after 5, 21 and 35 days for histological analysis.

Results

Scaffold characterisation revealed the porous structure, organic and amorphous content. This biomaterial promoted the adhesion, spreading and in vitro viability of the BMMSCs. Osteogenic-supplemented media did not improve the cellular response compared to DMEM. The biomaterial presented high biocompatibility and slow biodegradation in vivo. Remains of biomaterial were still observed at 21 and 35 days after implantation. However, on the 21st day, alveolar bone and epithelial healing were completely established.

Conclusions

These results indicate that chitosan-gelatin support the adhesion and osteogenic differentiation of rat BMMSCs and offer adequate physico-chemical and biological properties for use as scaffolds in bone tissue engineering-related strategies.     

数字化载葡萄糖酸氯己定PLA/nHA复合支架的制备及性能分析

目的:探讨负载葡萄糖酸氯己定壳聚糖纳米球(CSn-CG)3D打印多孔聚乳酸/纳米羟基磷灰石(PLA/nHA)支架的理化性能、细胞相容性及抗菌活性。方法:借助离子交联法,完成CSn(空白壳聚糖纳米球)与CSn-CG的制备,借助透射电镜(TEM)对纳米球形态进行观察。利用数字化设计3D打印技术,以PLA和nHA为原料,制备PLA/nHA支架。以浸泡法搭载CSn、CSn-CG,实验分为PLA/nHA组、PLA/nHA/CSn组、PLA/nHA/CSn-CG组3组。针对各组支架,通过扫描电镜(SEM)、X射线光电子能谱仪(XPS)、傅立叶红外光谱仪(FTIR)、X射线衍射(XRD)进行表征分析。体外释放实验评估支架的缓释性能,CCK-8法评估支架的生物相容性,琼脂扩散法检测支架的抗菌效果。采用SPSS25.0软件包对所得数据进行统计学分析。结果:CSn-CG为大小均匀的纳米球。SEM下发现,各组支架均为三维网状结构,孔径规则。体外释放结果表明,CG能够自支架内低速缓慢释放,用时30 d。CCK-8结果显示,PLA/nHA/CSn-CG组可促进小鼠前体成骨细胞(MC3T3-E1)增殖。体外抑菌实...     

拔牙后牙槽窝即刻植入纳米羟基磷灰石的临床观察

目的:研究拔牙后即刻植入纳米羟基磷灰石颗粒预防术后牙槽骨吸收,保持牙槽嵴高度的临床疗效。方法:选择16例需同时拔除下颌两侧相同部位后牙的患者,按左右分组,左侧为拔牙后立即填塞颗粒型纳米羟基磷灰石,右侧为拔牙后传统搔刮血块充盈对照。4周、12周分别复诊,摄X线片检查。对牙槽窝的愈合,牙槽嵴高度进行观察。结果:16例患者创口愈合良好。两组比较,12周后实验组X线片见牙槽窝处的X线阻射影与周围牙槽骨密度相近,界限不清,恢复的牙槽嵴与周围基本平齐,牙槽高度恢复良好。对照组牙槽嵴高度明显降低,实验组牙槽嵴高度降低不明显。结论:拔牙创内即刻植入纳米羟基磷灰石不影响创口愈合,能促进新骨的形成,很好地维持牙槽嵴高度,为以后进行义齿修复提供一个良好的基骨条件。     

麦角硫因对过氧化氢诱导的成骨细胞损伤的保护作用

目的 探讨麦角硫因(ergothioneine, EGT)对过氧化氢(H₂O₂)诱导的成骨细胞损伤的保护作用及机制。方法 用H₂O₂刺激MC3T3-E1细胞建立氧化应激模型，在加入不同浓度的EGT后，采用CCK8、流式细胞术分别检测细胞活力及凋亡率，利用荧光探针法和WST-8法检测细胞内活性氧(ROS)水平和总SOD酶活性，利用Western blot和RT-qPCR实验分别检测抗氧化及成骨相关蛋白和基因的表达水平，利用碱性磷酸酶染色对细胞成骨能力进行检测。结果 EGT可以剂量依赖地提升氧化损伤环境下MC3T3-E1细胞的存活率，降低H₂O₂引起的ROS生成以及Nrf2、CAT和HO-1的基因和蛋白表达。进一步发现EGT能拮抗H₂O₂对细胞成骨分化的抑制作用。结论 EGT可以抑制H₂O₂引起的氧化应激损伤，并进一步缓解因氧化损伤导致的成骨分化抑制。     

丝素蛋白/纳米羟磷灰石支架的实验研究

目的设计和构建三维丝素蛋白/羟磷灰石骨组织工程支架材料。方法联合运用丝素蛋白非编织方法和仿生矿化技术,制备并表征三维多孔丝素蛋白/纳米羟磷灰石的有机/无机组织工程支架。结果仿生矿化在非编织支架上形成的针状羟磷灰石晶体,直径20～60 nm,长100～300 nm。复合支架孔隙度为70%～78%,孔径为（163.4±42.6）μm。结论采用非编织丝素蛋白和仿生矿化的方法可制备孔隙度和孔径可控的组织工程支架。     

Lipopolysaccharide derived from Aggregatibacter actinomycetemcomitans inhibits differentiation of osteoblasts

Background and objectiveAlveolar bone defects in aggressive periodontitis are generally caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) through osteoclast differentiation facilitated by the interaction between the host's immune system, stimulated by the bacterial outer components of A. actinomycetemcomitans, and osteoblasts. Recently, some reports on the direct effects of A. actinomycetemcomitans on osteoblast differentiation have been published. However, the mechanisms in detail have still remained unknown. We herein demonstrated that A. actinomycetemcomitans might inhibit differentiation of osteoblasts and identified a causative substance.Materials and methodsThe following culture supernatants of A. actinomycetemcomitans were added to mouse osteoblasts, MC3T3-E1: supernatants fractionated by the molecular weight, supernatant supplemented with proteinase K and then heated, and supernatant supplemented with polymyxin B. Subsequently, RNA was extracted to determine the expressions of bone differentiation markers, alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP). Additionally, lipopolysaccharide (LPS) was added to MC3T3-E1 culture to measure the markers.ResultsA. actinomycetemcomitans culture supernatant, added to the MC3T3-E1, inhibited the expressions of bone differentiation markers at all concentrations. Only the fraction with a molecular weight of >100 kDa inhibited the bone differentiation. Neither proteinase K nor heating had an effect. However, polymyxin B completely abrogated the differentiation inhibitory activity. A. actinomycetemcomitans LPS concentration dependently inhibited the expressions of the bone differentiation markers.ConclusionsA. actinomycetemcomitans LPS suppressed osteoblast differentiation. This suggests that suppressed bone differentiation is involved in the alveolar bone destruction.     

成骨诱导剂对拔牙创愈合的影响

目的 探讨以明胶海绵为载体,地塞米松、维生素C和β-甘油磷酸钠组成的成骨诱导剂对拔牙创愈合和牙槽嵴形态改建的影响。方法选用50只家兔,拔除双侧上颌第一前磨牙,右侧拔牙创内填入载有成骨诱导剂的明胶海绵,作为实验侧;左侧填入空载明胶海绵,作为对照侧。拔牙后第1、2、4、8、12周各处死10只动物,取双侧牙槽骨标本,拍摄X线片,并测量骨缺损区新骨密度;用组织学方法评价拔牙创愈合情况;并于12周时,测量拔牙区牙槽嵴高度吸收值。结果X线片骨密度测量显示：术后2、4、8、12周,实验侧骨密度值均高于对照侧,差异有统计学意义（P<0.01）。组织学检查显示：实验侧拔牙创内成骨现象较对照侧早,成骨细胞分化和增殖更活跃。
12周时实验侧牙槽嵴高度吸收值小于对照侧,差异有统计学意义（P<0.01）。结论 由地塞米松、β-甘油磷酸钠和维生素C组成的成骨诱导剂能促进拔牙创愈合,加速成骨和骨改建。     

Effect of Nano-hydroxyapatite and platelet-rich fibrin covered by the amniotic membrane on osseointegration after mandibular piezoelectric ridge splitting

ObjectiveThis study aimed to assess the clinical and radiographic findings obtained by using amniotic membrane (AM) to cover nano-hydroxyapatite (nHA) bone grafts coated with platelet-rich fibrin (PRF) and thereby evaluate the osseointegration of posterior mandibular implants inserted simultaneously during alveolar piezoelectric ridge splitting technique (RST).MethodsA prospective cohort study was implemented with thirty patients who had a narrow posterior mandibular alveolar ridge and required implant restoration. Patients were distributed randomly into three groups (group I treated by piezoelectric RST and immediate implant insertion, augmented by the nHA bone graft only; group II treated by piezoelectric RST augmented by nHA bone graft and covered by AM; while group III was treated by piezoelectric RST augmented with PRF and nHA graft and covered by AM). Patients were evaluated clinically to assess the implant stability quotient (ISQ) and radiographically to assess horizontal ridge dimension, crestal bone level (CBL), and bone densitometric (BD) parameters.ResultsISQ results showed a non-significant clinical difference between groups while CBL values showed a high statistically significant difference over the 12-month interval when comparing groups III and II with group I. BD outcomes showed statistically significant differences at all intervals in comparisons of group III with groups I and II.ConclusionsThe results of this study suggest that concomitant use of PRF with nHA graft covered with AM for augmentation around the dental implant in a narrow posterior mandible after piezoelectric alveolar ridge splitting accelerate osseointegration and significantly increase bone density around the osseointegrated implant and decrease bone resorption in comparison to that achieved with the graft alone.     

Demineralised human dentine matrix stimulates the expression of VEGF and accelerates the bone repair in tooth sockets of rats

ObjectiveIn this study we investigated the possible use of human demineralised dentine matrix (DHDM), obtained from the extracted teeth, as bone graft material and evaluated the expression of vascular endothelial growth factor (VEGF) induced by this material in the healing process of tooth sockets of rats.DesignTo evaluate bone regeneration and expression of VEGF induced by DHDM, thirty-two male Wistar rats weighing approximately 200 g were used. After maxillary second molar extraction, the left sockets were filled with DHDM and the right sockets were naturally filled by blood clot (control). The animals were sacrificed at 3, 7, 14 and 21 days after surgery and upper maxillaries were processed for histological, morphometric and immunohistochemical analyses. DHDM was used to evaluate the mechanical effect of bone graft material into sockets. Expression of VEGF was determined by immunohistochemistry in all groups.ResultsOur results demonstrated a significant increase in the newly formed bone tissue in sockets of 7, 14 and 21 days and a significant increase in VEGF expression at days 7 and 14 on treated sockets.ConclusionsOur results showed that DHDM increases the expression of VEGF and accelerates the healing process in rats tooth sockets, by stimulating bone deposition and also vessels formation. These results suggest that DHDM has osteoinductive/osteoconductive potential and may represent an efficient grafting material on guided bone regeneration.     

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.     

The repair of critical-size defects with porous hydroxyapatite/polyamide nanocomposite: an experimental study in rabbit mandibles

This study was conducted to evaluate the healing of critical-size surgical defects after implantation of porous nano-hydroxyapatite/polyamide composite (nHA/PA) blocks based on a bilateral mandible model using adult New Zealand white rabbits. 15 rabbits were divided randomly into three groups according to the observation period: 4, 12 and 24 weeks. The defects on one side were implanted with nHA/PA blocks and the contralateral defects were kept empty as blank controls. A combination of macroscopic, radiographic, histological and histomorphometric studies were performed up to 24 weeks postoperatively and compared with normal healing. Large amounts of callus and active osteoblasts were found in the pore structure after 4 weeks of implantation, and the defects were completely occupied by neo-bone with density comparable with that of host bone at 24 weeks. Significant difference was found between nHA/PA groups and blank controls regarding X-ray opacity over the whole period and bone parameters at 4 weeks postoperation (P < 0.05). The porous nHA/PA composite promotes bone formation over the extension of the defect, particularly in the early stage. Porous nHA/PA offers interesting potential for maxillofacial reconstructive procedures in load-free areas.