【作者】 梅芳；

【导师】 刘鸿亮； 杨小平；

【作者基本信息】 北京化工大学 ， 材料科学与工程， 2010， 博士

【摘要】 人们投入巨大的努力来制造各种生物材料来满足某些特殊的临床需要。本研究中,我们将聚左旋乳酸(poly(L-lactic acid), PLLA)、多壁碳纳米管(multiwalled carbon nanotubes, MWNTs)和羟基磷灰石(hydroxyapatite, HA)混悬液通过电纺工艺制造出一种新型的引导性组织再生膜和组织工程支架材料。MWNTs/HA纳米颗粒在膜中分布均匀,膜的降解性能得到了很大的改善。细胞学实验证实,与对照组PLLA膜相比,在PLLA/MWNTs/HA膜上,牙周膜细胞(periodontal ligament cells, PDLCs)粘附和增殖增加了30%,而牙龈上皮细胞(gingival epithelial cells, GEC)则减少了30%。将PDLCs接种于PLLA/MWNTs/HA膜上,并将二者的复合体植入裸鼠腿部的肌袋中。组织学检查结果显示贴附在膜上的PDLCs在体内功能良好。这种新型膜材料虽然只是单层结构,但是却表现出良好的双重生物学功能,能够很好的满足引导性组织再生术的要求。与市场上正在出售的GTR膜或正在研究的GTR膜相比,这种膜可以简化生产步骤,降低生产费用并避免了临床应用过程中可能出现的错误。而且,这种膜在术后不需要去除。PLLA/MWNTs/HA膜在GTR和组织工程方面拥有巨大的应用潜力。本研究初步探索人羊膜基质细胞(human amniotic mesenchymalcells, hAMC)作为骨组织工程种子细胞的潜力。对人羊膜基质细胞进行分离培养,将第3代PDLCs在含有地塞米松(0.1μmol/L)、维生素C(50 mg/L)和β-磷酸甘油钠(10mmol/L)的DMEM培养液进行成骨诱导培养1周,进行茜素红染色,计数钙化结节形成数量,并进行免疫荧光细胞化学染色以检测Ⅰ型胶原和碱性磷酸酶的表达。利用免疫组织(细胞)化学染色检测羊膜及hAMC中FasL的表达。结果显示人羊膜基质细胞经过成骨诱导后,可见明显钙化结节形成,平均每孔18个。钙化结节处细胞Ⅰ型胶原和碱性磷酸酶表达阳性。人羊膜组织中细胞及体外培养的hAMC均可检测到FasL的阳性表达。这提示人羊膜基质细胞有潜力成为一种较为理想的骨组织工程的种子细胞。本研究探索了人羊膜基质细胞和电纺聚乳酸／羟基磷灰石(Poly (L-lactic acid)/hydroxyapatite, PLLA/HA)膜构建骨组织工程细胞／支架复合体。分离培养羊膜基质细胞,利用MTT法检测PLLA和PLLA/HA膜浸提液对羊膜基质细胞增殖的影响；将第3代羊膜基质细胞与PLLA和PLLA/HA膜在成骨诱导培养液中复合培养,在第1周和第4周时进行组织学检查,并在第4周时进行免疫荧光细胞化学染色以检测Ⅰ型胶原和碱性磷酸酶的表达。结果显示PLLA和PLLA/HA膜浸提液对羊膜基质细胞均无明显细胞毒性。羊膜基质细胞与两种膜材料复合培养后,细胞增殖明显,而且可以观察到钙化结节的形成,钙化结节处细胞Ⅰ型胶原和碱性磷酸酶表达阳性。这表明人羊膜基质细胞与电纺聚乳酸／羟基磷灰石膜可以共同构建成细胞／支架复合体,具有应用于骨组织工程的潜力。综上所述,本研究中制作的电纺丝膜具有良好的生物相容性,骨组织修复相关细胞在电纺丝膜上可以较好的行使功能。更多还原

【Abstract】 Significant effort has been devoted to fabricate various biomaterials to satisfy specific clinical requirements. In this study, we developed a new type of guided tissue regeneration (GTR) membrane by electrospinning of suspension consisted of poly (L-lactic acid)/multi-walled carbon nanotubes/hydroxyapatite (PLLA/MWNTs/HA). MWNTs/HA nanoparticles were uniformly dispersed in the membranes and the degradation characteristics were far improved. Cytologic researches revealed that the PLLA/MWNTs/HA membrane enhanced the adhesion and proliferation of periodontal ligament cells (PDLCs) by 30% and inhibited the adhesion and proliferation of gingival epithelial cells (GECs) by 30% also compared with control group. After PDLCs were seeded into the PLLA/MWNTs/HA membrane, cell/membrane composites were implanted into the leg muscle pouches of immunodeficiency mice. Histologic examinations showed PDLCs attached on the membranes functioned well in vivo. This new type membrane shows excellent dual biological functions and satisfied the requirement of GTR technique successfully in spite of a monolayer structure. Compared with other GTR membranes on sale or in research, the membrane can simplify the manufacturing process, reduce the fabrication cost and avoid possible mistakes in the clinical application. Moreover, it needs not to be taken out after surgery. PLLA/MWNTs/HA membranes have shown great potentials for GTR and tissue engineering.We also investigated the potential of human amniotic mesenchymal cells served as seeding cells in bone tissue engineering. Human amniotic mesenchymal cells (hAMC) were isolated, cultured. The third passage of hAMC was cultured in osteogenic induce media (DMEM supplemented with 10% FBS,0.1μmol/L dexamethasone,50 mg/L ascorbic acid and 10 mmol/L P-glycerophosphate) for 1 week. Calcified nodules were shown by alizarin red staining and counted under light microscope. Immunofluorescence cytochemical staining was used to detect COL I and ALP. Expression of FasL was examined in the amnion and hAMC by immunohistochemistry or immunocytochemistry. After osteoblast differentiation, calcified nodules were formed, average 18 per well. hAMC in calcified nodules were shown positive expression of COL I and ALP. FasL was detected positive both in cells contained in amnion and hAMC. It is concluded that human amniotic mesenchymal cells were potential ideal candidates for seed cells in bone tissue engineering.We constructed seed cells/scaffold complex by human amniotic mesenchymal cells (hAMC) and electrospun poly (L-lactic acid)/hydroxyapatite (PLLA/HA) membranes for bone tissue engineering. Human amniotic mesenchymal cells were isolated and cultured. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of PLLA and PLLA/HA membranes on the proliferation of hAMC. The third passage of hAMC were seeded on PLLA and PLLA/HA membranes respectively and cultured in osteogenic induce media (DMEM supplemented with 10% FBS,0.1μmol/L dexamethasone,50 mg/L ascorbic acid and 10 mmol/Lβ-glycerophosphate). Histological examination and HE staining was done at 1 and 4 week, and immunofluorescence cytochemical staining was done to detect COL I and ALP at 4 week. MTT assay showed that there was no significant difference between PLLA, PLLA/HA and control group during 7 days (P>0.05). After seeded onto two kinds of membranes, hAMC proliferated actively and calcified nodules were observed. Cells in calcified nodules were shown positive expression of COL I and ALP. It is concluded that Human amniotic mesenchymal cells and PLLA/HA can construct cells/scaffold complex and show great potential of application in bone tissue engineering.更多还原