表面修饰化明胶海绵支架与颌下腺种子细胞复合培养的研究

目的 应用表面修饰技术处理的明胶海绵与颌下腺种子细胞复合培养,研究表面修饰技术对颌下腺种子细胞在支架材料上附着、生长和分泌功能的影响.方法 选用明胶海绵36块,随机分A、B、C 3组,每组12块.分别用单纯明胶海绵、层粘连蛋白表面修饰的明胶海绵材料、转化生长因子β3表而修饰的明胶海绵材料与鼠颌下腺细胞在体外复合培养.培...     

表面修饰化明胶海绵支架与颌下腺种子细胞复合培养的研究术

目的应用表面修饰技术处理的明胶海绵与颌下腺种子细胞复合培养,研究表面修饰技术对颌下腺种子细胞在支架材料上附着、生长和分泌功能的影响。方法选用明胶海绵36块,随机分A、B、C3组,每组12块。分别用单纯明胶海绵、层粘连蛋白表面修饰的明胶海绵材料、转化生长因子B3表面修饰的明胶海绵材料与鼠颌下腺细胞在体外复合培养。培养后第3、7、15天行组织学观察、扫描电镜观察,第15天行免疫组织化学鉴定细胞来源,测定上清淀粉酶含量。结果组织学和扫描电镜观察可见培养后第3天各组细胞分散于材料表面,无细胞突起形成;第7天B组细胞数量明显多于A组和c组,细胞突起形成并锚定于胶原海绵表面;第15天B组细胞数量仍多于A组和C组,并可见细胞之间有触突联系和腺管样结构形成。免疫组织化学染色观察复合培养的颌下腺细胞BrdU呈强阳性。随着接种后培养时问的延长,各组颌下腺细胞分泌的淀粉酶含量均有不同程度的增加。第15天C组淀粉酶含量明显高于A组和B组（P〈0．05）。结论应用层粘连蛋白和转化生长因子β3对明胶海绵进行表面修饰,可促进颌下腺种子细胞在材料上的附着、增殖和分泌功能。     

TGF-β3对体外培养颌下腺细胞影响的研究

目的：研究转化生长因子β3（transforming growth factor β3,TGF-β3）对鼠颌下腺细胞（rat submandibular glandcells,RSGCs）的生长和分化的影响。方法：原代及传代培养RSGCs并观察TGF-β3对其增殖、分化和淀粉酶分泌功能的影响。结果：0.5ng/ml～10.0ng/mlTGF-β3明显促进颌下腺细胞的分化和分泌功能,对细胞的增殖无抑制作用。结论：TGF-β3能促进RSGCs的分化和淀粉酶的分泌功能,但对细胞的增殖无明显影响。     

层粘连蛋白对体外培养颌下腺细胞影响的研究

目的：研究层粘连蛋白（laminin，LN）对鼠颌下腺细胞（rat submandibular gland cells，RSGCs）生长及分泌功能的影响。方法：取对数生长期的第2代大鼠颌下腺细胞用于实验，按加入不同浓度LN分4组（0、5．0、25．0和50．0mg／L）进行培养，相差显微镜的观察，绘制生长曲线，MTT比色法、自动生化分析仪检测LN对细胞增殖及淀粉酶分泌功能的影响。结果：培养的RSGCs免疫组化鉴定CK8．13、S-100蛋白染色呈阳性，波形丝蛋白染色呈阴性。培养72h后MTT法检测5．0-50．0mg／LLN组细胞吸光度A值与对照组比较差异有统计学意义（P〈0．05）。培养96h后检测培养液中淀粉酶的含量，各浓度组与对照组相比差异无统计学意义（P〉0．05）。结论：LN能促进RSGCs的黏附和增殖并保持RSGCs的正常分泌功能。     

SD大鼠原代颌下腺细胞体外培养的生物学特征研究

目的：研究SD大鼠颌下腺细胞体外培养的生物学特征,为组织工程化涎腺样组织的体外构建提供有功能的种子细胞。方法：获取新生SD大鼠幼崽颌下腺组织,经酶消化后体外培养,倒置显微镜、电镜下行形态学及免疫细胞化学染色观察。绘制生长曲线,对涎腺细胞的生物特征进行评价。结果：体外培养的颌下腺细胞CK(anti-cytokeralin)及淀粉酶染色阳性,体外增殖活跃。结论：体外培养的颌下腺细胞增殖活跃,保持了腺细胞的生物特征,可作为体外构建组织工程化人工涎腺研究的种子细胞。     

异丙肾上腺素对SD大鼠原代颌下腺细胞增殖的影响

目的研究异丙肾上腺素对颌下腺细胞增殖的影响,为体外构建组织工程化涎腺提供充足的种子细胞。方法采用不同的方式加入异丙肾上腺素后,用Brdu标记阳性的细胞比率及绘制生长曲线的方法,检测颌下腺细胞的增殖能力。结果体外培养条件下,颌下腺细胞保持了对肾上腺素能受体刺激的反应,但与给药浓度及方式有关。结论异丙肾上腺素能在一定作用时间和浓度条件下促进颌下腺细胞的增殖,能为体外构建组织工程化涎腺提供种子细胞。     

大鼠下颌下腺细胞培养及其生物学特性研究

目的:体外培养大鼠下颌下腺细胞,并对其生物学特性进行研究,为涎腺疾病的探索提供体外模型。方法:无菌条件下取7 d龄Wistar大鼠双侧下颌下腺,仔细分离脂肪、包膜、神经和血管,采用组织块培养法进行培养。运用酶消化法和差速贴壁法纯化细胞;免疫组化SP法检测Cytokeratin-8(CK-8)抗体和α-Amylase抗体的表达;PAS染色法检测细胞糖原分泌;扫描电镜(scanning electronic microscopy,SEM)观察细胞的形态学特征。结果:组织块培养法可成功获得下颌下腺细胞,免疫组化染色可见大部分细胞胞质为棕黄色,提示CK-8抗体表达阳性,α-Amylase抗体表达阳性;PAS染色可见胞质呈紫红色;SEM可见细胞伸出长短不一的伪足,胞核着色深,有些细胞可见分泌颗粒。结论:组织块培养法可以成功培养大鼠下颌下腺细胞,并且操作简便。     

组织工程化口腔黏膜支架材料的研究进展

随着组织工程技术的发展,近年来国内外许多学者致力于口腔黏膜组织工程的研究,取得了一定的成就,支架材料是黏膜组织工程的一个重要因素,本文对组织工程化口腔黏膜的支架材料研究作一简要综述。     

应用人恒牙牙髓干细胞与明胶支架构建组织工程骨的研究

目的构建由人恒牙牙髓干细胞和三维明胶支架组成的组织工程骨,并验证其成骨效果。方法从正畸治疗减数拔除的恒前磨牙牙髓组织中应用酶消化法获得牙髓细胞,单抗Stro-1标记、免疫磁珠阳性分选系统分选获得牙髓干细胞。第3代细胞被分别接种到6孔板或三维明胶支架上进行成骨向诱导培养,诱导14天后支架组移植入裸鼠背部皮下。应用ALP染色、Von Kossa染色验证牙髓干细胞的体外成骨能力;应用X线、HE染色和免疫组化验证组织工程骨的体内成骨效果。结果牙髓干细胞体外成骨向诱导后7、14天ALP染色呈阳性;14、21天Von Kossa染色形成矿化结节。在体内组织工程骨成骨明显,X线显示实验侧支架内有团块状高密度阻射影;HE染色显示大量的骨组织形成;免疫组化显示骨结构区域骨桥蛋白、骨涎蛋白和骨钙素阳性表达。结论人恒牙牙髓干细胞和明胶支架构建形成了组织工程骨。     

利用RCCS体外快速构建组织工程化骨的实验研究

目的:探讨利用旋转式细胞培养系统(RCCS)在体外快速构建组织工程骨的可行性。方法:将骨髓基质细胞接种于煅烧骨鄄胶原支架上,分别在RCCS和静态环境中进行培养,8、24和72h后取材,细胞计数,检测碱性磷酸酶活性,扫描电镜观察成骨潜能。结果:随培养时间延长,细胞增殖和碱性磷酸酶活性均有所增长,但RCCS组明显高于静态培养组。超微结构观察,可见有颗粒状分泌物和丝状胶原纤维,形成三维结构。结论: 旋转式细胞培养系统是体外快速构建组织工程化骨的理想方法。     

新型自组装肽组织工程支架的研究进展

自组装肽组织工程支架是由互补的两性寡肽组成的纳米级并具有自组装特性的一类新型支架。其纳米级的微观结构与天然细胞外基质类似,这一特性使其成为一种真正的三维细胞培养支架。因此,这类支架具有一些其他支架所无法比拟的优势和特点。下面就自组装肽组织工程支架的结构、特点和应用作一综述。     

Cellular reactions to polylactide-based sponge and collagen gel in subcutaneous tissue

Polylactide copolymer and collagen are now used as bio-absorbable scaffold materials for restoration of lost oral tissues. Polylactide caprolactone (PLCL) sponge and collagen gel were examined for their cellular reactions when implanted in 8-week-old Sprague-Dawley (SD) rats' subcutaneous tissues for up to 8 weeks. The PLCL sponges were slowly absorbed by a mild chronic inflammation process in which multinucleated giant cells covered and slowly captivated the sponge surfaces without thick encapsulation. On the other hand, collagen gel was surrounded by thick inflammatory reaction zones and infiltrated by acute inflammation cells including neutrophils, lymphocytes and macrophages, and disappeared more rapidly. Although the bio-absorption process differed, both materials, when combined, appear to be useful scaffold materials for tissue engineering therapy in future dental practice.     

Application of collagen hydrogel/sponge scaffold facilitates periodontal wound healing in class II furcation defects in beagle dogs

Kosen Y, Miyaji H, Kato A, Sugaya T, Kawanami M. Application of collagen hydrogel/sponge scaffold facilitates periodontal wound healing in class II furcation defects in beagle dogs. J Periodont Res 2012; 47: 626–634. © 2012 John Wiley & Sons A/S Background and Objective: A three‐dimensional scaffold may play an important role in periodontal tissue engineering. We prepared bio‐safe collagen hydrogel, which exhibits properties similar to those of native extracellular matrix. The aim of this study was to examine the effect of implantation of collagen hydrogel/sponge scaffold on periodontal wound healing in class II furcation defects in dogs. Material and Methods: The collagen hydrogel/sponge scaffold was prepared by injecting collagen hydrogel, cross‐linked to the ascorbate‐copper ion system, into a collagen sponge. Class II furcation defects (of 5 mm depth and 3 mm width) were surgically created in beagle dogs. The exposed root surface was planed and demineralized with EDTA. In the experimental group, the defect was filled with collagen hydrogel/sponge scaffold. In the control group, no implantation was performed. Histometric parameters were evaluated 2 and 4 wk after surgery. Results: At 2 wk, the collagen hydrogel/sponge scaffold displayed high biocompatibility and biodegradability with numerous cells infiltrating the scaffold. In the experimental group, reconstruction of alveolar bone and cementum was frequently observed 4 wk after surgery. Periodontal ligament tissue was also re‐established between alveolar bone and cementum. Volumes of new bone, new cementum and new periodontal ligament were significantly greater in the experimental group than in the control group. In addition, epithelial down‐growth was suppressed by application of collagen hydrogel. Conclusion: The collagen hydrogel/sponge scaffold possessed high tissue compatibility and degradability. Implantation of the scaffold facilitated periodontal wound healing in class II furcation defects in beagle dogs.     

组织工程研究中的免疫学问题

组织工程学利用生命科学和工程学的原理及方法修复已破坏的组织与器官,这为患者带来了新的希望。然而,仍有一些问题限制了组织工程的临床化。其中,宿主对移植物产生的免疫排斥现象就是亟待解决的问题之一。现将组织工程研究中的免疫学问题作一综述。     

Clinical and histological evaluation of subepithelial connective tissue after collagen sponge implantation in the human palate

Rocha AL, Shirasu BK, Hayacibara RM, Magro‐Filho O, Zanoni JN, Araújo MG. Clinical and histological evaluation of subepithelial connective tissue after collagen sponge implantation in the human palate. J Periodont Res 2012; 47: 758–765. © 2012 John Wiley & Sons A/S Background and Objective: Successful root‐coverage treatment depends on the thickness of the donor tissue. This study aimed to evaluate the thickness of donor tissue after augmentation of the connective tissue in the palatal area by implantation of lyophilized collagen sponge (Hemospon^®). Material and Methods: Ten patients with an indication for root coverage, whose palate was deficient in adequate connective tissue, were recruited. The procedure was carried out in two stages. In the first stage, the palatal thickness in the donor site was measured at three standardized points (points 1, 2 and 3), from the distal of the canine to the distal of the first molar, and the lyophilized collagen sponge was inserted. In the second stage, the palatal thickness over the implant was measured (at points 1, 2 and 3), two biopsies of the palatal mucosa were collected – one over the implant (experimental sample) and the other on the contralateral side (control sample) – and then root‐coverage treatment was performed. Analyses consisted of clinical assessment of the palatal measurements before and after sponge implantation, and histological assessment of the experimental and control biopsy samples. Data were analyzed using the Wilcoxon test. Results: Both analyses showed a significant increase in mean thickness, of 1.08 mm of neoformed tissue in the clinical analysis (the tissue at point 2 was the thickest of the three points) and of 0.53 mm in the histological analysis. Conclusion: The insertion of lyophilized collagen sponge induced a significant increase in the thickness of palatal connective tissue.     

Bio-Oss胶原结合骨髓基质细胞构建组织工程骨的实验研究

目的:研究Bio-Oss胶原作为骨组织工程支架材料的可行性。方法:抽取成年小型猪骨髓,贴壁法获得骨髓基质细胞(BMSCs),经成骨诱导培养液体外培养、扩增、诱导后观察细胞增殖情况,并进行I型胶原、骨钙素的免疫细胞化学检测。将培养的第3代细胞接种于Bio-Oss胶原,进行超微结构观察,并将Bio-Oss胶原/BMSCs复合物植入裸鼠背部皮下,4、8周后进行组织学检测。结果:当接种密度为0.8×106/ml时,细胞与支架材料之间有最大附着量;VonKossa染色可见钙结节形成;I型胶原、骨钙素免疫细胞化学染色呈阳性;超微结构观察可见细胞生长附着于材料网孔内表面;组织学检测提示,8周时复合物内有新骨形成。结论:Bio-Oss胶原/BMSCs复合物显示成骨活性,Bio-Oss胶原可用作骨组织工程支架材料。     

以Bio-oss胶原为支架材料的组织工程化骨修复种植体周围骨缺损的初步实验研究

目的研究以Bio-oss胶原为支架材料的组织工程化骨修复种植体周围骨缺损的效果。方法取4只小型猪设立自身对照,获取骨髓基质细胞(BMSCs),经成骨诱导培养液体外培养、扩增、诱导后进行I型胶原、骨钙素的免疫细胞化学检测,将培养的第3代细胞同Bio-oss胶原构建BMSCs/Bio-oss胶原复合物,构建小型猪下颌骨种植体周围骨缺损模型,将复合物植入骨缺损处,通过影像学、能谱分析种植体-新骨界面Ca2+含量及组织学检测初步评价其促成骨作用。结果Ⅰ型胶原、骨钙素免疫细胞化学染色呈阳性,Von Kossa染色可见钙结节形成;超微结构观察可见细胞生长附着于材料网孔内表面;8周时成骨组织学观察可见BMSCs/Bio-oss胶原复合物植入组成骨量大于Bio-oss胶原植入组,能谱分析显示,2组Ca2+含量有统计学差异。16周时,组织学观察可见2组成骨量已无明显差别,能谱分析显示,2组Ca2+含量无统计学差异。结论利用BMSCs/Bio-oss胶原复合物修复小型猪种植体周围骨缺损相对于植入Bio-oss胶原能够缩短种植体发生骨结合的时间。     

一种新型纳米羟基磷灰石材料应用于牙周组织工程的可行性研究

目的:对一种新型纳米羟基磷灰石材料 -纳米羟晶 -胶原仿生骨材料 (nHAC)进行细胞相容性和细胞毒性的研究,以初步探讨其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(PDLCs)接种于nHAC三维支架上复合培养,采用细胞计数和扫描电镜观察PDLCs在nHAC支架上的附着、生长情况,并通过MTT测试法和碱性磷酸酶(ALP)活性检测法研究nHAC浸提液对PDLCs增殖和功能表达的影响。结果:人PDLCs在nHAC支架上形成良好贴附并增殖,扫描电镜可见nHAC具有良好的多孔网状结构,细胞在nHAC上生长旺盛,伸展充分,而PDLCs在不同浓度的材料浸提液中的生长、增殖及ALP活性与空白对照组相比无显著性差异。结论:nHAC具有良好的三维空间结构和细胞相容性,且无细胞毒性,有望成为牙周组织工程的支架材料。