Objective To construct the recombinant adeno-associated virus vector with human bone morphogenetic protein 4 gene（AAV-hBMP4）. Methods The hBMP-4 gene primer was designed basing on the corresponding gene sequence in GenBank. EcoR I site was introduced into the upstream of the primer and Sal Ⅰ site into downstream. The hBMP-4 gene was amplifiedwith the template of EX-A0242-M01-hBMP-4, then was cloned into pUC18 vectorto construct recombinant plasmid pUC18-hBMP-4. The plasmids pUC18-hBMP-4 and plasmid pSNAV cut by EcoR Ⅰ and Sal Ⅰenzyme, the fragments were collected and linked with T4 DNA ligase at 16℃ over night, recombinant plasmid pSNAVhBMP-4 was obtained. The recombinant plasmid was then transfected into BHK21 cells using Lipofectamine TM2000. The G418 resistant cells were obtained consequently. Thesecells were infected with HSV1-rc/△UL2 which has the function of packaging andcopying the recombinant AAV. After purification, the construction of recombinant AAV-hBMP-4 was completed. Results The construction of the recombinant pSNAV-hBMP-4 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-hBMP-4 wascorrect. The virus titer was about 1.5×1012 μg/ml.The purity of the virus was more than 95% using the SDSPAGE method. Conclusion With this method, high virus titers and purity of AAV-hBMP-4 can be acquired successfully and it is useful to bone tissue engineering.
Objective To evaluate the effects of sacrectomy extent on the stabil ity of lumbo-il iac fixation using single or dual il iac screw technique, and to determine which conditions require the dual il iac screw technique. Methods Nine fresh L2 to pelvic specimens were harvested from donated adult cadavers. After testing the intact state simulated by L3-5 pedicle screw fixation, sequential partial sacrectomies and L3 to il iac fixation using bilateral single il iac screw (Single) wereconducted on the same specimen as follows: in group A, under S1 partial sacrectomy and Single; in group B, under 1/2 S1 partial sacrectomy and Single; in group C, one-side (left) sacroil iac joint resection and Single; in group D, total sacrectomy and Single; and in group E, the single il iac screw was replaced by dual il iac screws based on group D. Biomechanical testing was performed on a material testing machine under 0-800 N compression and —7-7 N•m torsion loading modes for construct stiffness evaluation. Results The compressive stiffness of intact condition was (392 ± 119) N/mm, groups A, B, C, D, and E obtained 106.4% ± 9.5%, 102.7% ± 8.0%, 92.2% ± 10.1%, 72.7% ± 8.0%, and 107.7% ± 10.7% of intact condition, respectively. No significant differences were found among groups A, B, C, and the intact state (P gt; 0.05), however, the four groups showed significantly higher compressive stiffness than group D (P lt; 0.05). Although group E exhibited a comparable compressive stiffness with groups A, B, and intact state (P gt; 0.05), it displayed markedly higher compressive stiffness than groups C and D (P lt; 0.05). The torsional stiffness of intact state was (3.22 ± 1.23) N•m/deg. Groups A, B, C, D, and E acquired 105.4% ± 10.1%, 89.8% ± 12.3%, 75.9% ± 10.6%, 71.2% ± 10.2%, and 109.1% ± 16.9% of intact state, respectively. No significant differences were detected among groups A, B, E, and the intact state (P gt; 0.05). However, groups C and D showed remarkably lower torsional stiffness than groups A, E, and the intact state (P lt; 0.05). Importantly, group E offered remarkably higher torsional stiffness than group B (P lt; 0.05). Conclusion After under 1/2 S1 partial sacrectomy, single il iac screw technique could effectively restore local stabil ity; whereas it could hardly provide adequate stabil ity for further resection of one-side sacroil iac joint or total sacrectomy; in such situation, the use of dual il iac screw technique could obtain sufficient construct stabil ity. Therefore, in the surgical treatment of sacral tumor, the dual il iac screw technique should be considered for the unstable conditions of totalsacrectomy or under 1/2 S1 sacrectomy with one-side sacroil iac joint resection.
Objective To compare the maximum pull-out strength of the upper il iac screw and lower il iac screw with and without polymethylmethacrylate (PMMA) augmentation, and to provide the experimental evidences for the rational use of il iacscrews. Methods Ten intact human il ium from 5 donated cadavers with formal in embalmed were selected. The bone mineral density (BMD) of L1-4 of each cadaver was measured with a dual energy X-ray absorptiometry. The screws placed in the upper and lower il iac column were named as the upper and lower il iac screw, respectively. Using 70 mm length and 7.5 mm diameter screws with and without PMMA augmentation, 4 il iac screw technique models were sequentially establ ished and tested as follows: upper il iac screw (group A), upper il iac screw with PMMA augmentation (group B), lower il iac screw (group C), and lower il iac screw with PMMA augmentation (group D). Each il ium was mounted on a material testing machine with its position similar to standing. Under 2 000 cycl ic compressive loadings of 100-300 N to the screw, the maximum pull-out strength of il iac screw was measured. Results The BMD value of the 5 human cadavers was (0.88 ± 0.06) g/cm2. All the il iac screws were inserted into the screw tracts accurately as expected. No screw penetrations of acetabulum or cortex was not observed through visual inspection. There was no “halo” ring sign surrounding any screw after the 2 000 cycle loading. The maximum pull-out strengths of groupsA, B, C, and D were (964 ± 250), (1 462 ± 266), (1 537 ± 279), and (1 964 ± 422) N, respectively. Group D exhibited the highest maximum pull-out strength among the 4 groups (P lt; 0.05). No significant difference was detected between groups B and C (P gt; 0.05); however, groups B, C showed higher maximum pull-out strength than group A (P lt; 0.05). Conclusion The lower il iac screw offers significantly higher fixation strength than the upper il iac screw; PMMA augmentation could effectively increase the fixation strength of il iac screws and therefore could be appl ied in the salvage of il iac screw loosening.