Objective To investigate the effects of flow shear stress and mass transport on the construction of largescale tissue engineered bone using a perfusion bioreactor. Methods Bone marrow (20 mL) was harvested from the il iac crestof the healthy volunteer, and then hBMSCs were isolated, cultured and identified. The hBMSCs at passage 3 were seeded on the critical-size β-TCP scaffold and cultured in a perfusion bioreactor for 28 days. Different flow shear stress (1 ×, 2 × and 3 ×) and different mass transport (3, 6 and 9 mL/min) were exerted on the cells seeded on the scaffold by changing the viscosity of media or perfusion flow rate. The cell prol iferation and ALP activity of cells seeded on the scaffold were detected, and histology observation and morphology measurement of cell/scaffold complex were conducted. Results When the perfusion flow rabe was 3 mL/min, the cell viabil ity of 2 × group was higher than that of other groups (P lt; 0.05). When the flow shear stress was 3 ×, no significant differences were found among 3, 6 and 9 mL/min in cell viabil ity (P gt; 0.05). When the perfusion flow rate was 3 mL/min, the activity of ALP of 2 × and 3 × groups was higher than that of 1 × group (P lt; 0.05). When the flow shear stress was 3 ×, the activity of ALP of 6 mL/min group was the highest (P lt; 0.05). After 28 days of perfusion culture, the ECM of all the groups distributed throughout the scaffold, and the formation and mineral ization of ECM was improved with the increase of flow shear stress when the perfusion flow rate was 3 mL/min. However, the increase of perfusion flow rate decreased the mineral ization of ECM when the flow shear stress was 3 ×. Conclusion As two important fluid dynamics parameters affecting the construction of large-scale tissue engineered bone, the flow shear stress and the mass transport should be measured duringthe process of constructing large-scale tissue engineered bone so as to maximize their roles.
Objective To identify the expression of nerve growth factor (NGF) and its high affinity receptor (tyrosine kinase receptor A, TrkA) during bone induction by recombined human bone morphogenetic protein 2 (rhBMP-2) by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) and to discuss the role of NGF on the bone induction of the BMP. Methods Thirty-six ICR mice were divided into the experimental groupand the control group at random. rhBMP-2 /collagen sponge and collagen sponge were implanted into the right thigh muscle pouches of the mice in the experimental group and the control group, respectively. The tissues in the implanted site of the two groups were removed on the 7th, 14th and 21st day after the implantation. Histological, immunohistochemical and RT-PCR analyses were performed to detect osteoinductive effects of rhBMP-2 and the expression of NGF and TrkA. Results Gross observation showed that a solid lump was found in theright thigh in the experimental group on the 7th day and became harder on the 14th and 21st day, which was not found in the control group. rhBMP-2/collagen sponge displayed a potent ability to induce bone formation, while immunostaining for NGF and TrkA was observed in the course of osteoinduction by rhBMP-2. On the 7th day in the experimental group, NGF positive immunostaining reached the peak in thestage of chondrogenesis and there were a large number of cells expressing NGF, including fibroblasts, chondroblasts, chondrocytes, and osteoblasts; then, therewas a decrease in the number of the positivecells and in the intensity of immunostaining on the 14th and 21st day. Staining of TrkA wassimilar to that of NGF. The expression level of the mRNA of NGF during the course of bone induction peaked 7 days after the implantation and then decreased. Conclusion rhBMP-2/collagen is a kind of satisfactory osteoinductive material, and many different kinds of cells induced by rhBMP-2 can express NGF and TrkA, which suggests that NGF may play an important role in the osteogenesis initiated by exogenous BMP through direct and indirect pathways.
Objective To study the immunological tolerance induced by blocking the second signal of T cell with extrinsic cytotoxic T lymphocyteassociated antigen 4 immuno globlin(CTLA4-Ig). Methods Fifty-four BALB/C mice, inbred strains, were employed as recipients of bone allografts, using a model of heterotopic muscle pouch. The 54 mice were divided into 3 groups and18 for each group. The first group, in which the donor was C57BL/6 with intraperitoneal injection ofL6(as a control), was named AL group. The second group,also C57BL/6 with injection CTLA4-Ig, was named AC group. The third group,homologous BALB/C with injection of PBS buffer solution, was named AB group.The serum antibody, lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor, the analysis of lymphocyte subsets, a regraft experiment and histology were determined 2, 4 and 6 weeks after transplantation. The second transplantation was to regraft C57BL/6(BC group) and C3H(BHgroup) mice respectively after first 12 mice being transplantated with C57BL/6 and injected with CTLA4-Ig as to detect donor-specificity of immunological tolerance. Results Compared with AB group, AL group created more intensive immune rejection: CD4 T cell subsets(Plt;0.05), the serum antibody(Plt;0.05) and lymphocyteproliferation of the second stimulation by splenic cell and bone supernatant ofdonor (Plt;0.01 and 0.05) were significantly increased. However, the results of AC group showed that CTLA4-Ig significantly inhibited the immune rejection: CD4T cell subsets(Pgt;0.05), the serum antibody (Pgt;0.05), and lymphocyte proliferation of the second stimulation(Pgt;0.05) were similar to those of AB group. Histological observation of AC group showed that lymphocyte infiltration disappeared,cartilage and new bone formed, and bone marrow cavities emerged. A regraft experiment showed that CD4 T cell subsets (Plt;0.05) and lymphocyte proliferation of the second stimulation by splenic cell and bone supernatant of donor(Plt;0.05), BC group was significantly lower than those of BH group. So theimmunological tolerance induced by CTLA4-Ig was of donor-specificity. Conclusion The immunological tolerance induced by CTLA4-Ig was prolonged for 6 weeks. This study provides a brand-new path for bone transplantation, which can be helpful to other organ transplantation.
【Abstract】 Objective To evaluate the effectiveness of HA mixed with adenovirus mediated rhBMP-2 gene (AdvrhBMP-2) transferred BMSCs of goats on distraction osteogenesis. Methods Nineteen adult goats were used for the experiment,no matter they were male or female, and the weight of the goats were 15-20 kg. The 10 mL marrow was obtained from theil iac crest of each goat. The BMSCs was expanded and passaged conventionally. The 3th BMSCs was infected by Adv-rhBMP-2 at 200 multipl icity of infection (MOI). The 1×108 infected BMSCs were digested by 0.25% trypsin and collected, then mixed with HA. The right tibia lengthening models were developed, and mixture with BMSCs was injected in the osteotomy position. The goats were divided randomly into 4 groups according to the material injected in operation, group A: Adv-rhBMP-2/BMSCs/HA (n=6); group B: Adv-rhBMP-2/BMSCs (n=5); group C: Adv-β-gal/BMSCs/HA (n=4); group D: sham without any injections (n=4). After a seven-day latency period following ostectomy, distraction was carried out at a rate of 1 mm/day for 4 weeks. Roentgenography was practiced in 5, 8 and 12 weeks. After 12 weeks, the goats were sacrificed and dual-energy X-ray absorptiometry (DXA), biomechanical test and histology results were studied. Results After five and eight weeks surgery, X-raytest showed the distraction callus was more in group A and B than group C and D, and the radiographic score was significantly higher in group A and B than in the other two groups(P lt; 0.05); after 12 weeks surgery, the continued callus was formed in the distraction defects in all groups. DXA showed the mean bone mineral content of distraction callus in group A, B, C, D was (4.175 ± 1.921), (2.600 ± 0.638), (2.425 ± 0.826) and (1.175 ± 0.574) g, and the mean bone mineral density was (0.612 ± 0.196), (0.630 ± 0.159), (0.450 ± 0.166) and (0.266 ± 0.113) g/cm2. The group A and B was significantly higher than group C and D (P lt; 0.05).Biomechanical test showed the maximum loading of group A, B, C, D was (490.20 ± 155.08), (350.59 ± 80.48), (221.95 ± 68.79) and (150.65 ± 92.29) N, and elastic modulus was (178.24 ± 105.80), (105.88 ± 27.09), (81.18 ± 48.67) and (50.35 ± 47.64) MPa. The group A was significantly higher than in group C and D (P lt; 0.05). Histology observation revealed abundant bone formation in the distraction defects in group A, and the bone trabecula was arranged longitudinal and netl ike. Histomorphology analysis revealed the bone volume in group A, B, C, D was 72.35% ± 5.68%, 67.58% ± 7.42%, 49.63% ± 4.87% and 38.87% ± 2.35%, and the bone formation was significantly greater in group A compared with group D (P lt; 0.05). Conclusion HA mixed with rhBMP-2 modified BMSCs can accelerate distraction osteogenesis in goats.
To construct the retroviral vector containing human interleukin 1 receptor antagonist (IL-1Ra)and to investigate the property of the transfected articular chondrocytes from osteoarthritic patients in vitro. Methods Retroviral vector PLXRN carrying IL-1Ra (PLXRN-IL-1Ra) gene was constructed by inserting IL-1Ra gene at the sites of Sal I and BamH I. The recombinant retroviral plasmid was homologously recombinated in bacterial cells. After screening and ampl ification, the recombinant retroviral plasmid was obtained and transfected into PT67 cells. The repl ication-defective retrovirus PLXRN-IL- 1Ra was packed and ampl ified in the PT67 cells. Viral titer was determined by infecting NIH/3T3 cells with serially diluted viral supernatants produced with a control vector. Experiments were divided into 3 groups: non-transducted group (group A), PLXRN transduction group (group B), PLXRN-IL-1Ra transduction group (group C). Primary articular chondrocytes from osteoarthritic patients were transduced with PLXRN and PLXRN-IL-1Ra.The positive chondrocytes clones, which were G418- resistant, were cultured for 3-4 weeks after being selected by G418. The expression of IL-1Ra mRNA in the chondrocytes was determined by RT-PCR. Levels of IL-1Ra protein synthesis in the supernatants were measured by ELISA. Results Restric tive endonuclease identification and gene sequencing confirmed that the recombinant contained IL-1Ra cDNA．Virus titer could reach 3 × 104 CFU/mL. Primary chondrocytes cultured in vitro were polygonal or spindle and were stained with purple particles by toluidine blue staining. After stable transduction into the chondrocytes the 311 bp fragment of IL-1Ra was detected in group C by semi-quantitative RT-PCR. ELISA showed that IL-1Ra in supernatants of the group A and group B were below the level of detection. The concentrations were（60.47 ± 15.13）ng/L in group C .There were significant differences between gene transduction group and control groups (P lt; 0.05). Conclusion The construction of recombinant retrovirus vector by homologous recombination in bacterial cells can be quickly and easily performed. Stable and effective expression of IL-1Ra can be achieved by transduction with retroviral vectors in osteoarthritic articular chondrocytes, indicating potential util ity in gene therapy for osteoarthritis．