颌下腺细胞在不同孔隙率支架材料上相容性的研究

目的研究颌下腺细胞与支架材料体外复合培养中,支架材料的孔隙率对颌下腺细胞黏附、增殖及分化的影响.方法体外原代培养SD鼠颌下腺细胞,并传至第2代,将细胞浓度为50×106/ml的颌下腺细胞分别接种在孔隙率为76.3%、81.4%、90.6%、95.8%的胶原海绵支架上.于培养后1,3,5,7天取材,HE染色、扫描电镜观察及细胞计数检测等,评价何种孔隙率的胶原海绵支架适宜颌下腺细胞的增殖与分化.结果 SD鼠颌下腺细胞在孔隙率76.3%及81.4%的支架上增殖、分化较慢;在孔隙率为90.6%及95.8%的支架上生长良好,增殖较快,细胞周围分泌较多细胞外基质.结论颌下腺细胞在孔隙率为90%～95%的支架材料上,形成的细胞--支架复合物较佳.     

颌下腺细胞在不同孔隙率支架材料上相容性的研究

目的　研究颌下腺细胞与支架材料体外复合培养中 ,支架材料的孔隙率对颌下腺细胞黏附、增殖及分化的影响。方法　体外原代培养SD鼠颌下腺细胞 ,并传至第 2代 ,将细胞浓度为 5 0× 1 0 6/ml的颌下腺细胞分别接种在孔隙率为 76 .3%、81 .4 %、90 .6 %、95 .8%的胶原海绵支架上。于培养后 1 ,3,5 ,7天取材 ,HE染色、扫描电镜观察及细胞计数检测等 ,评价何种孔隙率的胶原海绵支架适宜颌下腺细胞的增殖与分化。结果　SD鼠颌下腺细胞在孔隙率 76 .3%及 81 .4 %的支架上增殖、分化较慢 ;在孔隙率为 90 .6 %及 95 .8%的支架上生长良好 ,增殖较快 ,细胞周围分泌较多细胞外基质。结论　颌下腺细胞在孔隙率为 90 %～ 95 %的支架材料上 ,形成的细胞支架复合物较佳。     

表皮生长因子对颌下腺细胞在胶原海绵黏附的影响

目的　探讨表皮生长因子 (EGF)对颌下腺细胞在胶原海绵支架上黏附的影响。方法体外原代培养SD鼠颌下腺细胞 ,并传至第 2代 ,将其分别接种于含表皮生长因子修饰的胶原海绵上 ,和不含表皮生长因子修饰的胶原海绵上。于接种后 7d取材 ,进行细胞计数计算细胞贴附率 ;苏木素 伊红 (HE)染色 ,扫描电镜观察等检测。结果　实验组含有EGF ,细胞贴附率为 73 .1% ,对照组不含EGF ,贴附率为 62 .8% (P <0 .0 5 ) ,HE染色及扫描电镜观察也证实 :实验组的颌下腺细胞在胶原海绵支架上黏附 ,增殖及分化均优于对照组。结论　EGF对颌下腺细胞在胶原海绵上的黏附有促进作用。     

组织工程颌下腺细胞与胶原海绵复合培养的实验研究

目的研究大鼠颌下腺细胞在胶原海绵材料支架上的生长情况。方法取8d龄Wistar大鼠颌下腺细胞，传代培养至第2代细胞，按2×10^4／ml注射法接种于5mm×5mm×2mm胶原海绵表面进行培养。术后1、2、3周行组织学观察、扫描电镜观察胶原海绵上接种颌下腺细胞形态结构特点；取复合培养3周的颌下腺细胞行免疫组织化学细胞角蛋白（cytokratin8．13，CK8．13）、α-平滑肌肌动蛋白（α-smooth muscular actin，α-SMA）染色，透射电镜观察细胞超微结构，鉴定细胞来源。分别取复合培养1d，1、2、3周的培养上清，用Amano法测定淀粉酶含量，检测与胶原海绵复合培养的颌下腺细胞功能。结果细胞接种后第1周细胞分散于材料表面，无细胞突起形成；第2周细胞数量有所增加、细胞突起形成并锚定于胶原海绵表面；第3周时附着的细胞数目增多，可见细胞表层有丝状纤维形成。免疫组织化学染色观察复合培养的颌下腺细胞上皮细胞特异性抗体CK8。13呈强阳性，肌上皮细胞特异性抗体α-SMA染色呈阳性。透射电镜下颌下腺上皮细胞表面可见微绒毛、胞浆皱褶和细胞顶部的酶原颗粒，胞核大卵圆形，胞质内见线粒体和粗面内质网。随着接种后培养时间的延长，与胶原海绵复合培养的颌下腺细胞分泌的淀粉酶含量有不同程度的增加。结论胶原海绵具有良好的细胞相容性，颌下腺细胞与胶原海绵复合培养，细胞可保持增殖分化能力及分泌功能，有望成为颌下腺细胞的载体应用于组织工程支架材料。     

两种支架材料与颌下腺细胞体外复合培养的比较研究

目的将颌下腺细胞分别与颌下腺脱细胞基质(SGAM)生物衍生材料及胶原海绵体外复合培养,比较两种不同支架材料的生物相容性。方法用传代培养第2代的颌下腺细胞分别与SGAM生物衍生材料及胶原海绵体外复合培养,在一定时间通过扫描电镜观察细胞在不同支架材料上的生长情况。结果颌下腺细胞在SGAM生物衍生支架材料上,多数附着在支架材料的孔隙内或其边缘,数量较多,大小不等,形态各异,表面凸凹不平,突起丰富;表面结构似“银耳”状。而细胞在胶原海绵支架材料上大多附着在材料表面,数量较少,突起较少。结论颌下腺脱细胞基质支架材料与胶原海绵支架材料相比具有良好的生物相容性,是一种较为理想的颌下腺组织工程用支架材料。     

胶原海绵为载体构建大鼠羊膜上皮细胞复合体的实验研究

目的：大鼠羊膜上皮细胞（AECs）体外培养并种植于胶原海绵形成细胞支架复合体后,探讨细胞在支架上分别于体内及体外时的生长情况。方法：取第三代AECs细胞种植于胶原海绵支架。光镜下及用荧光标记法观察细胞种植前后的增殖情况;ELISA法检测细胞支架上清液中VEGF、bFGF、TGFβ的含量。并通过荧光标记示踪法观察细胞支架植于体内后细胞的存活情况。结果：AECs在胶原海绵支架上生长情况良好,细胞支架上清液中VEGF、bFGF、TGFβ的含量随时间增长呈不断上升趋势,将细胞支架移植于体内后经过观察发现细胞仍存活良好。结论：AECs在胶原海绵支架上及体内移植后生长情况良好,这为利用AECs胶原海绵支架复合体进行细胞治疗奠定了实验基础。     

胶原海绵与兔软骨细胞体外三维培养的实验研究

目的 观察用人胎盘Ⅰ型胶原海绵作为兔软骨细胞体外三维培养支架时，软骨细胞形态学特征和功能及该支架的吸附性和组织相容性。方法 将新生兔原代和传代软骨细胞接种于人胎盘Ⅰ型胶原海绵进行体外培养，用光镜和扫描电镜观察细胞形态和结构及支架的吸附性和组织相容性，用免疫组化观察细胞Ⅱ型胶原合成。结果 以胶原海绵作为支架的传代软骨细胞能维持其形态学特征及细胞功能，该支架有较好的吸附性和组织相容性。结论 人胎盘Ⅰ型胶原海绵可作为软骨细胞体外三维培养较好的支架材料。     

胶原凝胶复合骨胶原基质(BCM)负载关节软骨细胞的实验研究

目的 探讨胶原凝胶包埋软骨细胞接种BCM支架的三维培养对软骨细胞生长及功能的影响.方法 将胶原凝胶包埋的关节软骨细胞接种BCM支架并在体外培养,应用倒置相差显微镜和扫描电镜观察软骨细胞的粘附、生长和增殖情况,培养14d,行苏木精-伊红、甲苯胺蓝染色观察软骨组织形成情况.结果 软骨细胞在支架上粘附、生长和增殖良好,体外培养14d能形成较成熟的软骨组织.结论 胶原凝胶复合BCM支架具有良好的细胞相容性,可作为负载生长因子的载体.     

增强型生物活性玻璃/胶原复合材料的促血管化作用

目的骨组织工程所使用的支架材料能否快速、有效地血管化是骨修复的关键。研究增强型生物活性玻璃/胶原复合材料对血管化的协同和促进作用,为构建血管化组织工程骨修复骨缺损寻找适宜的支架材料。方法体外分离获取人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),并通过血管性血友病因子(von Willebrand factor,vWF)与CD34抗原鉴定,取第1代细胞用于实验。将细胞接种于增强型生物活性玻璃/胶原复合材料上,扫描电镜观察细胞黏附情况。取细胞接种于增强型生物活性玻璃/胶原复合材料作为实验组,等量细胞直接接种于培养板常规培养作为对照组,采用alarmarBlue法动态检测细胞增殖率,实时荧光定量PCR检测内皮细胞相关基因VEGF、血管内皮生长因子受体1(fms-related tyrosine kinase1,Flt-1)、血管内皮生长因子受体2(kinase insert domain receptor,Kdr)的mRNA表达。取SD大鼠6只,将支架材料包埋于大鼠股内侧皮下,实验组采用增强型生物活性玻璃/胶原复合材料、中央轴向植入血管束,对照组采用非增强型生物活性玻璃/胶原复合材料,分别于植入5、10d取出,行冰冻切片并HE染色动态观察支架材料内部的血管化状态。结果分离的细胞通过形态学及vWF、CD34免疫荧光鉴定为HU VECs。扫描电镜示HUVECs在支架材料上黏附较好。HUVECs在实验组支架材料上增殖明显,在第3天后细胞增殖率开始高于对照组,11d达到增殖平台期时细胞数仍多于对照组(P<0.05)。实时荧光定量PCR检测示,培养3d实验组VEGF、Flt-1、Kdr的mRNA表达均显著高于对照组(P<0.05)。包埋大鼠皮下实验可见,实验组5、10d时植入的血管仍通畅,其周围新生血管随时间延长而增多,材料周缘可见宿主血管浸润生长,但新生血管稀疏;对照组仅有纤维组织生长,10d时材料已大部分降解,组织难以长入。结论增强型生物活性玻璃/胶原复合材料在大鼠体内外可促进血管化,可能适于作为构建血管化组织工程骨的支架材料。     

干细胞构建组织工程皮肤的研究进展

组织工程学是近年来细胞生物学、工程材料学和临床医学交叉发展起来的一门新兴学科。组织工程学最基本的思路是在体外将分离的种子细胞接种到具有一定空间结构的载体支架上,通过细胞与细胞之间的相互作用形成具有一定结构功能的组织或器官。从组织工程学的观点来看,人工皮肤主要有三类：①表皮替代物,即培养表皮片;②真皮替代物,即胶原凝胶、胶原海绵合成膜、     

Submandibular accessory salivary gland causing Warthin's duct obstruction

BACKGROUND: Submandibular masses are mostly secondary to sialolithiasis. Salivary gland tumors should be considered in the differential diagnosis. In this case report, an unusual cause of Warthin's duct obstruction caused by an accessory salivary gland tissue is presented. METHODS: Sialography revealed the submandibular accessory salivary gland. RESULTS: Submandibular gland excision was performed and histopathologic investigation showed the accessory salivary tissue, which was narrowing the Warthin's duct. CONCLUSIONS: In cases of a symptomatic submandibular accessory gland, excision extirpation of the submandibular gland and accessory salivary tissue should be undertaken.     

Submandibular gland mucocele: report of two cases.

Submandibular gland mucocele should be remembered in the differential diagnosis of swelling at the submandibular triangle. In the cystic lesion of the submandibular area, the biochemical analysis of aspirated material for amylase should be performed. The cases with submandibular gland mucocele should be treated by removing the lesion with both the submandibular and the sublingual glands.     

Idiopathic submandibular sialoceles in the neck.

A submandibular sialocele is a subcutaneous cavity containing saliva. The clinical and radiologic features of 3 patients with an idiopathic submandibular sialocele are presented. All 3 patients were males in their twenties. Submandibular sialocele presents as a soft cystic and compressible neck mass, with no history of previous trauma or diseases of the salivary gland. Computed tomography (CT) of the neck revealed a homogenous lesion with enhancing rim. The lesion appeared to be insinuating into the surrounding tissue. Excision of the sialocele, leaving the submandibular gland intact, was performed for the first patient. Recurrence of a neck mass occurred after 4 months. Complete excision of the sialocele with associated submandibular gland was subsequently performed. There was no recurrence after a follow-up period of 3 years. Excision of gland and sialocele was performed for the other 2 patients. There was no recurrence after a follow-up of 2 years and 10 months, respectively.     

Innervation and secretory function of transplanted human submandibular salivary glands

Free submandibular gland autotransplantation is used to treat absolute tear deficiency. Although disconnected from any peripheral innervation, most transplants show increasing secretion for years. We have evaluated the secretory activity and autonomic innervation of such transplants. Secretory activity of glands in response to parasympatholytics and parasympathomimetics was evaluated by Schirmer's test and Technetium scintigraphy. Submandibular gland tissue specimens taken before and after transplantation were examined histologically. Relative hypersecretion during the first postoperative week suddenly decreased but then slowly increased during the first postoperative year. Hypersecretion was significantly reduced by parasympatholytics while carbachol rapidly increased secretion. Histology of transplanted glandular tissue showed parenchymal atrophy. Cholinesterase-positive nerves were abundant and in a similar distribution to normal with scattered positive ganglion cells. Adrenergic axons were fewer than normal and irregularly distributed. Early hypersecretion may be due to release of neurotransmitters from degenerating terminal axons. This is followed by a period of minimal secretion during which hypersensitivity of acinar cells develops. With spontaneous reinnervation, secretion is accentuated by external sympathetic vasomotor adrenergic drive. This shows that submandibular glands can remain viable despite complete separation from their normal nerve supply and are capable of regaining a substantial secretory activity for years.     

肌源性干细胞与胶原海绵复合培养的实验研究

目的：研究成午大鼠肌源性干细胞纯化培养方法以及与胶原海绵的细胞相容性，探讨构建组织工程化肌性吊带的可行性。方法：取成年雌性SD大鼠前肢肱三头肌，用胶原酶和Dispase进行消化，采用差速贴壁的方法纯化肌源性干细胞并与胶原海绵体外复合培养，构建组织工程化肌源性干细胞／胶原海绵复合体。结果：成功地分离培养了成年大鼠的肌源性干细胞，肌源性干细胞在胶原海绵上生长良好、增殖迅速。结论：采用差速贴壁的方法可以纯化肌源性干细胞，肌源性十细胞与胶原海绵有良好的细胞相容性，本实验为构建组织工程化肌性吊带的研究打下基础。     

Human mesenchymal stem cell implantation and collagen modification as a tool for tissue engineering

Tissue engineering, in all its aspects, is a focus of increasing interest. Optimum vascularization has to be ensured ahead of transferring experimental designs to clinical applications. Our group searched for matrix modifications and cell sources liable to increase vessel formation in engineered tissue. Notwithstanding the ethical issues, adult human mesenchymal stem cells (hMSC) seemed to offer as unlimited a possibility of proliferation and differentiation as embryonic stem cells. These series of experiments focused on the differentiation capability of adult stem cells and organisation in modified collagen sponges. Adult mesenchymal stem cells were cultured to the second generation. Two different groups were formed: Group A cells remained in medium without additional growth factors. Group B cells were cultured in angiogenic differentiation medium. On day 21, the cells of both groups were detached and implanted into different collagen sponges: unmodified collagen sponges and EDC/NHS [1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS)] cross-linked and heparinized collagen scaffolds. Twenty-one days later, the sponges were prepared for the histological evaluation. After 3 weeks in culture, the cells of group B showed phenotypic endothelial cell characteristics and expressed von-Willebrand factor. Enhanced proliferation and invasion on cross-linked and heparinised matrices were obtained with both cell types (A and B). In addition, the stem cell derived endothelial cells formed organised structures throughout the scaffolds. Obtained data revealed the differentiation capability of adult MSC into endothelial cells and organisation in modified collagen matrices. This approach might lead to optimised vascular incorporation in tissue replacement.M. Markowicz, A. Heitland have contributed equally in this work.     

前脂肪细胞组织工程学的初步探讨

目的探讨胶原-透明质酸海绵支架与前脂肪细胞复合培养形成工程化脂肪组织的可行性。方法切取成年女性腹部皮下脂肪组织，经体外分离培养前脂肪细胞，与胶原-透明质酸支架混合，移植到裸鼠体内，未混合细胞的支架作为对照组。移植后4周和8周取材，对所取标本进行大体观察及厚度、高度测量和免疫组织化学检测。结果初步证实复合物植入后能形成黄色脂肪样组织。有新生血管形成，免疫组织化学检测证实脂肪细胞的存在及良好分布。结论胶原一透明质酸海绵支架可以作为组织工程技术应用研究中形成脂肪组织的支架，移植到裸鼠体内，在其皮下能形成脂肪样组织。     

Human bone marrow mesenchymal stem cells seeded on modified collagen improved dermal regeneration in vivo

In the correction of functional and aesthetic impairments, loss of soft connective tissue creates the need for adequate implant material. The reconstruction of defects resulting from radical excisions, trauma, or hereditary diseases has seen the use of combined grafts and flaps. With the aim of minimizing donor site morbidity, new methods have been evaluated. Because of a low rate of vascularization, with artificial dermal templates the take has only been poor. As shown in previous studies, improved angiogenetic potency and epidermal formation has been obtained in modified, cell-seeded collagen matrices. We have now investigated the suitability of adult bone marrow mesenchymal stem cells (hMSC) for soft tissue engineering. In this study, hMSC were isolated and expanded. Cells (10(6)) were seeded onto EDC cross-linked collagen sponges and implanted in 30 immunodeficient mice. Collagen sponges without cells were used as controls. The grafts were evaluated after 2 and 6 weeks. After explantation, macroscopic appearance, weights, and histology (scaffold degradation, cellularity, and invasion depth of the seeded cells) were all assessed. After 2 and 6 weeks in vivo, new vessels were found macroscopically on all cell-seeded collagen grafts. The control grafts appeared to be degraded with a lower rate of vessel ingrowth. In the experimental group, weight gain was significant after 2 and 6 weeks in vivo compared to the same grafts after 72 h in vitro, while weight increased only slightly in the control group. Histologically, populated scaffolds showed a high density of vascularization under a capsule. The control sponges showed single capillaries and a thicker capsule. Compared to the controls, cellularity (cells/field) was greater in cell-containing collagen grafts after 2 and 6 weeks. The results obtained demonstrate that in vitro cultured human mesenchymal stem cells seeded on modified collagen sponges may be able to act as a replacement for soft tissue.