OBJECTIVETo reconstruct mandibular defect using tissue engineering bone with bone marrow stem cells (BMSCs) cell sheets and investigate the effect of cell sheets on osteogenesis.

METHODSBMSCs were isolated with the method of density gradient centrifugation from canine and cultured. BMSCs were induced to differentiate to osteoblasts. BMSCs induced were fabricated to BMSCs cell sheets. The poly (lactic-co-glycolic acid) (PLGA) wrapped with cell sheets were implanted into the mandibular defect in the left side (experimental side). PLGA wrapped without cell sheets were implanted into the right side (control side) of mandibles. 16 dogs were evenly divided into 4 groups, and one group of them was executed in 4, 8, 12, 16 weeks for gross investigation and histological observation.

RESULTSThe osteogenesis of experimental side was better than that of control side. 16 weeks after implantation, most areas of the mandibular defect were replaced by fresh bone tissue. Compact bone similar to normal bone tissue formed in the lingual defect of mandible and had bony union with the bone stump. The optical density of the fresh bone in the experimental side was higher than that of the control side, there was a significant difference between the two methods (P<0.05). Plenty of lamellar bones formed in experimental side and Haversian system, as well as red marrow, were observed.

CONCLUSIONTissue engineering bone with the structure of lamellar bones can be formed by the technology of BMSCs cell sheets.

Animals ; Bone Marrow Cells ; Bone and Bones ; Dogs ; Lactic Acid ; Mandible ; Osteoblasts ; Osteogenesis ; Polyesters ; Polyglycolic Acid ; Polymers ; Tissue Engineering

Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.
Animals ; Antigens, Viral ; immunology ; Base Sequence ; Capsid Proteins ; immunology ; Cell Line ; Cysteine Endopeptidases ; genetics ; Epitopes ; genetics ; Foot-and-Mouth Disease ; immunology ; prevention & control ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Molecular Sequence Data ; Mutation ; Porcine respiratory and reproductive syndrome virus ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, Attenuated ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology