| 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.
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