离子化胶原作为牙周组织工程支架材料的实验性研究

目的：检测具有不同表面电荷性质的离子化胶原材料对体外培养的人牙周膜成纤维细胞粘附、生长增殖的影响。方法：MTT法检测人牙周膜成纤维细胞在不同支架（天然胶原、甲基化胶原、琥珀酰化胶原）上的粘附、生长增殖情况,并用扫描电镜进行观察。结果：MTT法：与天然胶原比较,甲基化胶原更有利于人牙周膜成纤维细胞粘附（P〈0.05）,琥珀酰化胶原则减弱了细胞粘附（P〈0.05）。结论：相对于不具有表面电荷性质的天然胶原,具有表面正电荷性质的甲基化胶原材料促进了人牙周膜成纤维细胞粘附、生长增殖,为新型牙周组织工程支架材料的研究提示了一种方向。     

人牙周膜细胞在透明质酸/胶原支架上的黏附与生长

目的研究人牙周膜细胞（PDLC）在胶原、透明质酸及透明质酸/胶原支架上的黏附、生长情况,以初步探讨透明质酸/胶原支架应用于牙周组织工程的可行性。方法将体外培养的人牙周膜细胞接种到碳化二亚胺交联的胶原、透明质酸及透明质酸/胶原支架上;MTT法检测支架对人牙周膜细胞黏附、生长的影响;并用倒置相差显微镜和扫描电镜观察形态变化。结果MTT结果显示胶原、透明质酸及透明质酸/胶原支架上人PDLC的黏附、生长情况,在第1、2、4天组间比较差异具有统计学意义（P<0.05）,第7天组间差异无统计学意义（P>0.05）,且人牙周膜细胞数量透明质酸/胶原组均高于对照组;人牙周膜细胞在支架上生长良好。结论相对于胶原支架和透明质酸支架,透明质酸/胶原支架更有利于人牙周膜细胞的黏附,提示该材料具备成为牙周组织工程理想支架材料的潜力。     

人牙周膜细胞在透明质酸朋交原支架上的黏附与生长

目的研究人牙周膜细胞（PDLC）在胶原、透明质酸及透明质酸／胶原支架上的黏附、生长情况,以初步探讨透明质酸／胶原支架应用于牙周组织工程的可行性。方法将体外培养的人牙周膜细胞接种到碳化二亚胺交联的胶原、透明质酸及透明质酸／胶原支架上;MTT法检测支架对人牙周膜细胞黏附、生长的影响：并用倒置相差显微镜和扫描电镜观察形态变化。结果MTT结果显示胶原、透明质酸及透明质酸／胶原支架上人PDLC的黏附、生长情况,在第1、2、4天组间比较差异具有统计学意义（P〈0．05）,第7天组间差异无统计学意义（P〉0．05）,且人牙周膜细胞数量透明质酸／胶原组均高于对照组;人牙周膜细胞在支架上生长良好。结论相对于胶原支架和透明质酸支架,透明质酸／胶原支架更有利于人牙周膜细胞的黏附,提示该材料具备成为牙周组织工程理想支架材料的潜力。     

hBMP-2修饰β-TCP/I型胶原复合材料对MC3T3-E1细胞增殖的影响

目的:探究含人的骨形成蛋白-2为目的基因的质粒DNA修饰纳米β-磷酸三钙(Tricalcium phosphate)/I型胶原溶液复合材料对小鼠前成骨细胞增殖的影响。方法:含hBMP-2为目的基因的质粒DNA修饰纳米β-TCP/I型胶原溶液复合材料,建立MC3T3-E1细胞株与纳米复合材料体外培养体系。将其分为支架组和平皿组,扫描电镜、光镜观察细胞学形态,Alamar Blue法检测细胞增殖,MuseTMCell Analyzer法检测细胞周期,实时荧光定量PCR检测Cdk2、Cdk4等细胞增殖基因。结果:复合材料细胞黏附数、增殖数及表面分布高于平皿组(P<0.05),实验结果显示支架组均优于平皿组。结论:复合材料与单纯含hBMP-2为目的基因的质粒DNA相比, 更能促进前成骨细胞黏附、增殖及功能代谢, 表明其对前成骨细胞具有良好细胞相容性,可成为一种新型的具有应用前途的骨修复或组织工程材料。     

人牙髓干细胞在不同HA含量的PLGA/HA复合支架材料上的粘附研究

目的:构建不同质量构成比的PLGA/HA复合支架并评价材料机械性能;探讨不同比例PLGA/HA复合生物学支架对牙髓干细胞粘附能力的影响。方法:采用溶液浇筑/颗粒沥析技术构建出分别含有10%HA、20%HA及30%HA的PLGA/HA复合支架,对照组采用单纯PLGA支架。应用电子万能测验机检测支架的拉伸强度,扫描电镜观察表面结构。将牙髓干细胞与不同比例的PLGA/HA支架复合培养,应用DAPI染色细胞计数法检测细胞粘附能力。结果:在对3组实验组的拉伸强度测试中,10% HA组拉伸强度最高,20% 组次之,两组均高于对照组,而30% HA组拉伸强度低于对照组(P<0.05)。扫描电镜观察支架表面,显示支架孔径在100~250 μm之间,孔隙间 连通性良好,孔隙率较高。细胞接种于支架2、6、12 h后,荧光染色显示细胞与3种支架紧密贴合,粘附良好,细胞计数结果显示各实验组细胞粘附数量均多于对照组,其中在30%HA组中细胞粘附性能最佳(P<0.05)。结论:3种不同构成比的HA/PLGA复合支架均为良好的组织工程支架材料,其中10%HA组具有良好的机械性能,30%HA组细胞粘附性能更佳。     

京尼平交联改性可溶性蛋壳膜蛋白冻干支架的制备及检测

目的：评价京尼平交联改性前后可溶性蛋壳膜蛋白（soluble eggshell membrane protein,SEP）冻干组织工程支架的理化性能和生物相容性。方法：冷冻干燥法制备SEP的冻干支架,浸泡于京尼平溶液中交联改性。通过扫描电镜观察其表面形貌。测量其孔隙率、抗拉强度以及降解率,采用溶血实验、亚急性全身毒性实验和细胞毒性实验初步评价其生物相容性。结果：京尼平交联改性前后的SEP冻干支架孔径分别为（280±71）μm和（263±89）μm,孔隙率分别为（90.4±7.6）%和（87.9±9.7）%;交联改性显著提高了SEP冻干支架的拉伸强度和弹性模量,降低了支架的失重率（P〈0.01）;交联前后的材料均无溶血现象;亚急性短期全身毒性实验中组织切片均未见病理变化;细胞毒性检测均为0级。结论：京尼平交联改性在提高SEP冻干支架的力学强度和抗降解能力的同时,仍可保持支架材料良好的生物相容性。     

国产胶原膜加工改良及理化特性分析

目的 :本研究为了解决国内现有的胶原膜降解过快的问题 ,欲对其按双抗胶原膜的制作方法进行加工改良以更符合GTR要求 ,同时验证双抗胶原膜与普通胶原膜相比在理化性能方面的优越性和其原理的有效性。方法 :购天津产牛腱胶原膜、加工改良 ,并以原牛腱胶原膜为对照 ,检测理化性能指标弹性模量、膨胀率及体外酶降解的改变。结果 :牛腱胶原膜经加工改良后弹性模量由 (5 4 .77± 15 .4 6 ) g/mm2 增加到 (16 8.6 0± 2 0 .38) g/mm2 ,膨胀率由 4 .190± 0 .195降到 3.6 97± 0 .16 2 ,体外胶原酶降解时间由十余小时提高到 5～ 6d ,而制成双抗胶原膜后 ,则可 14d内也不被降解。结论 :双抗胶原膜的制作方法可应用于成品胶原膜的加工 ;加工改良后的双抗胶原膜具有较高的交联程度 ,较强的抗酶降解能力 ,其形态结构特点及理化性能与牛腱胶原膜相比较更符合GTR技术要求     

大鼠骨髓基质细胞促进自体骨形成的实验研究

目的研究自体骨髓基质细胞(bone marrow stromal cells,BMSCs)体外诱导扩增与仿生纳米壳聚糖-胶原复合支架材料(nano chitosan sodium collagen scaffold,NCSCS)复合修复骨缺损的可行性。方法分离纯化Wistar大鼠BMSCs,诱导BM-SCs向成骨细胞(osteoblast,OB)转化,经碱性磷酸酶、茜素红组织化学染色鉴定,将BMSCs复合仿生纳米壳聚糖-胶原复合支架材料(nano chitosan sodium collagen scaffold,NCSCS)进行扫描电镜观察。制作Wistar大鼠胫骨骨缺损模型,缺损处NCSCS-BMSCs复合物移植后观察骨缺损修复成骨情况。结果扫描电镜下BMSCs细胞在仿生纳米壳聚糖-胶原复合支架材料上大面积生长。BMSCs复合仿生纳米壳聚糖-胶原复合支架材料植入2周后,材料周围可见大量纤维组织,并可见成骨细胞和少量新生的骨样基质,新生骨对照组和实验组比较差异显著(P<0.01);植入6周后,可见到材料与新生骨之间相互混杂,中有纤维相间隔,和原有骨界面尚清晰可辨认,材料部分降解,对照组和实验组比较新生骨有统计学差异(P<0.05);植入12周后,材料几乎降解完全,和天然骨组织界面可见新骨形成,对照组和实验组比较新骨形成无统计学差异(P>0.05)。材料降解在各时间段对照组和实验组比较差别不显著(P>0.05)。结论 BMSCs介导NCSCS修复骨缺损优于单纯的NCSCS修复骨缺损,尤以早期效果为好。     

两种沟槽钛片上纤维连接蛋白修饰方法的效果比较

目的:比较三氟代乙烷磺酰氯与硅烷化两种钛片上修饰纤维粘结蛋白(Fibronectin,Fn)方法的效果。方法:通过三氟代乙烷磺酰氯与硅烷化将Fn修饰于微沟槽钛表面,XPS分析不同材料表面元素组成;荧光染色观察接种不同材料表面的蛋白数量及分布。DAPI染色检测HGF接种在不同材料表面2、4、6 h后早期附着细胞数。用cck8比较HGF接种不同材料表面6 h、12 h、1 d、3 d、5 d、7 d后细胞增值率。结果:XPS结果提示硅烷化组材料表面的纤维蛋白含量高于三氟组及空白组(P<0.05)。免疫荧光观察提示硅烷化组材料表面的纤维连接蛋白含量高于三氟组及空白组(P<0.05)。DAPI染色提示2 h时3组材料细胞附着数相当,4、6 h时硅烷化组材料表面上细胞数明显多于其他组材料(P<0.05)。CCK8结果提示硅烷化材料在6个观察时间段硅烷化组材料细胞增值率高于三氟组及空白组(P<0.05)。结论:硅烷化法在钛片上修饰纤维蛋白的效果优于三氟代乙烷磺酰氯的方法。     

丝素蛋白多孔支架用于口腔软组织增厚的初步研究

目的 通过体内实验探究3种不同浓度的丝素蛋白多孔支架在口腔软组织增厚应用中的可行性。方法采用冷冻干燥及甲醇增强法制备3种不同浓度：质量分数为1%(SF1组)、3%(SF3组)、5%(SF5组)的丝素蛋白支架,通过扫描电子显微镜（SEM）、傅里叶转换红外光谱（FTIR）、X射线衍射（XRD）、热重分析（DSC）对支架进行表征分析,并测定各组支架的孔径、孔隙率及体外降解速率。将3组支架材料（实验侧）与胶原基质（对照侧）分别植入新西兰大白兔两侧口腔黏膜下,比较其术前、术后3个月黏膜厚度的变化,通过组织苏木精-伊红（HE）染色法、Masson染色法对比观察各组材料的体内代谢及再生效果。结果 SEM显示：3组支架材料都是相互交联的多孔结构;XRD及FTIR表明：3组支架均以较稳定的SilkⅡ型结构为主,在体外降解较慢;其中SF3组的孔径最大（133.40μm±22.85μm）,孔隙率适中（90.05%±6.68%）。体内实验结果表明：除了SF1组因空间维持不足导致增厚效果类似于对照侧以外,SF3及SF5组的空间维持稳定、增厚效果明显优于对照侧;但不同于SF5组诱发了明显的炎症,SF3组体内降解较...     

Carbodiimide cross-linking inactivates soluble and matrix-bound MMPs, in vitro

Matrix metalloproteinases (MMPs) cause collagen degradation in hybrid layers created by dentin adhesives. This in vitro study evaluated the feasibility of using a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), to inactivate soluble rhMMP-9, as an example of dentin MMPs, and matrix-bound dentin proteases. The inhibitory effects of 5 EDC concentrations (0.01-0.3 M) and 5 incubation times (1-30 min) on soluble rhMMP-9 were screened with an MMP assay kit. The same EDC concentrations were used to evaluate their inhibitory effects on endogenous proteinases from completely demineralized dentin beams that were incubated in simulated body fluid for 30 days. Decreases in modulus of elasticity (E) and dry mass of the beams, and increases in hydroxyproline content of hydrolysates derived from the incubation medium were used as indirect measures of matrix collagen hydrolysis. All EDC concentrations and pre-treatment times inactivated MMP-9 by 98% to 100% (p < 0.05) compared with non-cross-linked controls. Dentin beams incubated in 0.3 M EDC showed only a 9% decrease in E (45% decrease in control), a 3.6% to 5% loss of dry mass (18% loss in control), and significantly less solubilized hydroxyproline when compared with the control without EDC cross-linking (p < 0.05). It is concluded that EDC application for 1 min may be a clinically relevant and effective means for inactivating soluble rhMMP-9 and matrix-bound dentin proteinases if further studies demonstrate that EDC is not toxic to pulpal tissues.     

丝素蛋白-软骨脱细胞外基质复合取向支架的制备及评价

目的 制备丝素蛋白（SF）-软骨脱细胞外基质（CECM）仿生支架材料,检测其理化性能特性。方法 采用改良温度梯度热诱导相分离（TIPS）技术结合冷冻干燥法制备出CECM取向支架,然后将CECM浆料与制备好的SF溶液按照质量比1∶1混合后配制成质量分数6%的浆料,通过TIPS技术制备出SF-CECM复合取向支架。对SF-CECM复合取向支架进行扫描电子显微镜（SEM）观察和组织学染色,同时测定支架孔隙率、吸水性以及力学性能。结果 SEM观察可见,支架横断面呈多孔网状结构,纵剖面呈垂直的管状结构。组织学染色显示复合支架脱细胞彻底,有蛋白多糖、胶原成分,与天然软骨成分相似。支架孔隙率、吸水率和纵向压缩弹性模量分别为95.733%±1.010%、94.309%±1.302%和（65.40±4.09）kPa。结论 SF-CECM复合取向支架具有良好的理化特性和生物力学性能,有望成为较理想的组织工程软骨支架。     

Growth and differentiation of mouse osteoblasts on chitosan-collagen sponges

The aim of this study was to investigate the effects of collagen on the microstructure and biocompatibility of chitosan-collagen composite sponges fabricated by a freezing and drying technique. The study was categorized into four groups: Group I: collagen; Group II: chitosan; Group III: 1:1 (by wt) chitosan-collagen and Group IV: 1:2 (by wt) chitosan-collagen sponges. A mouse osteoblast cell line, MC3T3-E1, was cultivated on the sponges in a mineralized culture medium for 21 days. Microstructure of scaffolds and growth of cells on the sponges were examined using scanning electron and confocal laser scanning electron microscopes. Pore size was analysed from scanning electron microscope images using Image-Pro Plus image analysis software. Cell viability (MTT assay), alkaline phosphatase activity and levels of osteocalcin and calcium were monitored every 3 days and on days 15 and 21, respectively. It was found that the sponges were porous with average pore sizes of 80-100 microm. A combination of chitosan and collagen matrixes created a well defined porous microstructure and biocompatible scaffolds. Chitosan-collagen composite sponges promoted growth and differentiation of osteoblasts into the mature stage. To optimize application of the composite sponges in bone regeneration, the fabrication process must be improved to increase the pore size of the scaffolds.     

拔牙位点保存用复合温敏凝胶的构建及体外表征

目的:制备多壁碳纳米管/羟基磷灰石/壳聚糖复合温敏凝胶,探讨其作为拔牙位点保存材料的理化性能。方法:采用原位沉积方法制备多壁碳纳米管/羟基磷灰石(MWNT/ HA简称HAC)复合无机材料,将其分散至壳聚糖温敏凝胶中,制备壳聚糖/多壁碳纳米管/羟基磷灰石(CS /MWNT/HA简称CS/HAC)复合温敏凝胶,以成胶时间、孔径、孔隙率、机械强度及降解性能为指标优化配方设计,并表征复合温敏凝胶的微观形貌及理化性能。结果:以10 g/L的HAC作为补强成分加入到壳聚糖溶液中,能够在不影响成胶并保证一定孔隙率(84%)的前提下,提高材料的机械性能(抗压强度0.441 MPa),所获复合温敏凝胶具有适宜的临床操作时间(12 min),并具有可控的降解速率。结论:负载HAC的复合温敏凝胶具有良好的成型性,并具有较好的机械性能,在拔牙位点保存领域具有良好的应用前景。     

Dentine collagen cross-linking using tiopronin-protected Au/EDC nanoparticles formulations

ObjectiveThe aim of this study was to investigate EDC-assisted collagen crosslinking effect with different concentrations of tiopronin-protected gold (TPAu) nanoparticles on demineralized dentine.MethodsTPAu nanoparticles were fabricated from 0.31-g tetrachloroauric acid and 0.38-g of N-(2-mercaptopropionyl) glycine (2.4-mmol). Then co-dissolved using 35-mL of 6:1 methanol/acetic acid and mixed using NaBH₄. EDC (0.3-M) was conjugated to TPAu nanoparticles at TPAU/EDC-0.25:1, and TPAU/EDC-0.5:1 treatment formulations ratios. Dentin specimens treated with 0.3-M EDC solution alone or left untreated were used as control. Nanoparticles formulations were characterized in term of particles morphology and size, Zeta potential, thermogravimetric analysis and small-angle X-ray scattering. Dentin substrates were characterized in term of TEM investigation, dentin proteases characterization, hydroxyproline liberation, elastic modulus measurement, Raman analysis and confocal microscopy viewing.ResultsTEM evaluation of tiopronin protected gold nanoparticles dispersion revealed nano-clusters formations in both groups. However, based on our TEM measurements, the particle-size was ranging from ˜20 to 50 nm with spherical core-shape which were almost similar for both TPAu/EDC ratios (0.5:1 and 0.25:1). Zeta potential measurements indicate negative nanoparticles surface charge. SAXS profiles for both formulations, suggest a typical profile for uni-lamellar nanoparticles. Superior dentin collagen cross-linking effect was found with the TPAu/EDC nanoparticles formulations compared to the control and EDC treated groups.SignificanceCross-linking of dentin collagen using TPAu coupled with EDC through TPAu/EDC nanoparticles formulations is of potential significance in improving the biodegradation resistance, proteases inhibition, mechanical and structural stability of demineralized dentin substrates. In addition, the cross-linking effect is dependent on TPAu/EDC ratio, whereas higher cross-linking effect was found at TPAu/EDC ratio of 0.5:1.     

Towards resorbable 3D-printed scaffolds for craniofacial bone regeneration

This review will briefly examine the development of 3D-printed scaffolds for craniofacial bone regeneration. We will, in particular, highlight our work using Poly(L-lactic acid) (PLLA) and collagen-based bio-inks. This paper is a narrative review of the materials used for scaffold fabrication by 3D printing. We have also reviewed two types of scaffolds that we designed and fabricated. Poly(L-lactic acid) (PLLA) scaffolds were printed using fused deposition modelling technology. Collagen-based scaffolds were printed using a bioprinting technique. These scaffolds were tested for their physical properties and biocompatibility. Work in the emerging field of 3D-printed scaffolds for bone repair is briefly reviewed. Our work provides an example of PLLA scaffolds that were successfully 3D-printed with optimal porosity, pore size and fibre thickness. The compressive modulus was similar to, or better than, the trabecular bone of the mandible. PLLA scaffolds generated an electric potential upon cyclic/repeated loading. The crystallinity was reduced during the 3D printing. The hydrolytic degradation was relatively slow. Osteoblast-like cells did not attach to uncoated scaffolds but attached well and proliferated after coating the scaffold with fibrinogen. Collagen-based bio-ink scaffolds were also printed successfully. Osteoclast-like cells adhered, differentiated, and survived well on the scaffold. Efforts are underway to identify means to improve the structural stability of the collagen-based scaffolds, perhaps through mineralization by the polymer-induced liquid precursor process. 3D-printing technology is promising for constructing next-generation bone regeneration scaffolds. We describe our efforts to test PLLA and collagen scaffolds produced by 3D printing. The 3D-printed PLLA scaffolds showed promising properties akin to natural bone. Collagen scaffolds need further work to improve structural integrity. Ideally, such biological scaffolds will be mineralized to produce true bone biomimetics. These scaffolds warrant further investigation for bone regeneration.     

Stabilization of dentin matrix after cross-linking treatments,in vitro

Objectives

To evaluate the effect of EDC on elastic modulus (E), MMPs activity, hydroxyproline (HYP) release and thermal denaturation temperature of demineralized dentin collagen.

Methods

Dentin beams were obtained from human molars and completely demineralized in 10 wt% H₃PO₄ for 18 h. The initial E and MMP activity were determined with three-point bending and microcolorimetric assay, respectively. Extra demineralized beams were dehydrated and the initial dry mass (DM) was determined. All the beams were distributed into groups (n = 10) and treated for 30 s or 60 s with: water, 0.5 M, 1 M or 2 M EDC or 10% glutaraldehyde (GA). After treatment, the new E and MMP activity were redetermined. The beams submitted to DM measurements were storage for 1 week in artificial saliva, after that the mass loss and HYP release were evaluated. The collagen thermal denaturation temperature (TDT) was determined by DSC analysis. Data for E, MMP activity and HYP release were submitted to Wilcoxon and Kruskal–Wallis or Mann–Whitney tests. Mass loss and TDT data were submitted to ANOVA and Tukey tests at the 5% of significance.

Results

EDC was able to significantly increase collagen stiffness in 60 s. 10% GA groups obtained the highest E values after both 30 and 60 s. All cross-linking agents decreased MMP activity and HYP release and increased TDT temperature. Significant differences were identified among EDC groups after 30 or 60 s of cross-linking, 1 M or 2 M EDC showed the lowest MMP activity.

Significance

Cross-linking agents are capable of preventing dentin collagen degradation. EDC treatment may be clinically useful to increase resin-dentin stability.