Table 1.
Transduction efficiency and cell pathological changes and optimal multiplicity of infection.
Table 2.
Sequence of primers.
Figure 1.
The demineralized bone matrix scaffold looked like white sponge with natural porous structure (A,B).
Figure 2.
An osteochondral defect (diameter = 6 mm; depth = 4 mm) generated in the central weight bearing surface of the femur condyle in knee(A). Prepared tissue-engineered scaffold were inserted into the defect(B).
Figure 3.
The trilateral and fibroblast-like cells were observed in primary cells 8 days after culture (A, Inverted phase contrast microscope×100). The primary BMSCs showed whirlpool growth 14 days after cultured (B, Inverted phase contrast microscope ×40).
Figure 4.
Multidirectional differentiation of BMSCs.
Culture of BMSC under adipogenic conditions induced drastic morphology changes including formation of cell aggregates and accumulation of lipid vacuoles in the cytoplasm which positive for oil red O staining. (A, ×200). BMSCs were induced into osteogenetic cells 14 days after osteogenetic induction. Alizarin red staining showed there was calcium nodules formation (B, ×40). There was pellet formation at the bottom of centrifuge tube after centrifugal. Glycosaminoglycan was detected using alcian blue staining in cartilage pellets at Day 21 of culture(C, white arrow points to the pellet). Presence of glycosaminoglycans was confirmed in histological sections of the pellet by alcian blue staining(D).
Table 3.
The flow cytometry results of surface markers from BMSCs in different generation.
Figure 5.
Adenoviral infection and the optimal multiplicity of infection.
The fluorescence expression of BMSCs after transduced with Ad- BMP-2 48h in MOI 10, 20, 50, 100 at 48 h(A,B,C,D),the fluorescence expression gradually strong with the increase of MOI. Transduction efficiency of Ad-BMP-2 was analyzed 48 hours post transduction by determining the population of cells with GFP expression within the total cell population(E,F). almost all the BMSCs express green fluorescence after transduced with Ad- BMP-2 48 h in MOI = 20(G). Transduction efficiency of Ad-TGF-β3 was conformed by flow cytometry, the Transduction efficiency was 95.1% 48 h post transduction in MOI = 50(H).
Figure 6.
Image of immunofluorescence, PCR electrophoresis and ELISA detection after transfection.
Image of immunofluorescence performed 72 hours after transfection of BMSCs. There was a positive red(A) or green(B) fluorescence staining in the BMSCs in B group or T group 72 h after transfection. Both the red and the green fluorescence were detected in the BT group (C). No positive red or green fluorescence was detected in the N group (D). PCR results showed a band at 310 bp (B group) and at 114 bp was detected in B and T group, respectively. In the BT group, both 310 bp and 114 bp bands were detected, and no band was detected in the control N group (E). ELISA assay showed that the expression of BMP-2 or TGF-β3 increased gradually and then peaked at 7 days following initial infection, and expression could still be detected till 21 days post-transfection (F,G).
Figure 7.
The expression of SOX-9, COL-2A, ACAN in B, T, BT group at 1W,2W,3W after transfection.
Total RNA from the samples of BMSCs in the B, T, BT, and N group at 7 d, 14 d and 21 d were extracted with RNeasy mini kit. Complementary DNA was prepared using SuperScript III first-strand synthesis system. Expression of SOX9, COL-II and ACAN were analyzed via iQ SYBRR green super mix on MyiQ single color Real Time polymerase chain reaction (RT-PCR) detection system. The relative expression expression of SOX-9, COL-2A and ACAN were significantly increased in BT group as compared with either of the individual treated groups (*P<0.05, * *P<0.01).
Figure 8.
Immunohistochemical image of COL-II 21 days after transfection.
Type II collagen expression was detected using a mouse monoclonal antibody (1∶200; Abcam, England) and a horseradish peroxidase-conjugated anti-mouse antibody (1∶50; Dako, Denmark), followed by colour development with diaminobenzidine tetrahydrochloride (DAB, Dako). The cell of BT group(C) acquired a strong type II collagen staining compared with B group (A) and T group (B). The N group (D) were negative for type II collagen staining.
Figure 9.
SEM image of DBM and the adhesion and morphology of induced BMSCs cultured on DBM.
SEM showed DBM had natural netlike structure with pores interconnected(A). A large number of cells was growing and covering the surface of DBM 2days after incubation(B). SEM showed the number of cells significantly increased(blue arrow), the spindled cell growing on the surface and the interior of DBM(red arrow)(C,D).
Figure 10.
The gross observation of group I, II, III, IV at operation, 4W, 8W and 12W after operation.
At 12 weeks post-operation, the full-thickness cartilage lesions had healed in I group, with the contour of the femur being nearly entirely repaired and both the color and quality of repaired tissue being similar to the surrounding healthy cartilage. In both the II and III groups, the defects did not appear to fully heal and remained irregular, with the contour of the femur surrounding the defects remaining visibly discernible. There was retained a severe degeneration, in IV group.
Figure 11.
The imagine of H&E and safranin O staining in group I, II, III, IV at 8W and 12W after operation.
It appears that the DBM scaffolds were gradually absorbed over time, and by 8 weeks post-operation they were completely absorbed in all groups. In the defect area, chondrocytes and cartilage lacunas were largely increased, and the repaired tissue were main hyaline cartilage with safranin O positive stain in I group. At 12 weeks post-operation, the repaired tissue was largely hyaline cartilage that stained positively with safranin O staining, as compared with other three groups.
Figure 12.
The The O'driscoll score of group I, II, III, IV at 8W and 12W after operation.
At 12W after implantation, I group scores were significantly higher than both control and non-treatment group scores (*P<0.05, * *P<0.01).