Effect of rAAV2-hTGFß1 Gene Transfer on Matrix Synthesis in an In Vivo Rabbit Disk Degeneration Model.

STUDY DESIGN: In vivo gene transfer for disk regeneration. OBJECTIVE: To evaluate the efficiency and effect of human transforming growth factor ß1 (hTGFß1) gene transfer mediated by adeno-associated virus (AAV) in a rabbit disk degeneration model induced by fibronectin fragment (Fn-f). SUMMARY OF BACKGROUND DATA: Gene therapy for disk degeneration has been reported to be effective. Nevertheless, few investigations have targeted the degenerative nucleus pulposus (NP) cells in vivo. Fn-f-induced degeneration has been previously verified to be a useful model for the study of disk degeneration at the molecular level. AAV vector is well suited for gene transfer in the disk for its lower immunogenicity and higher safety. MATERIALS AND METHODS: The early dedifferentiated NP cells were transfected with rAAV2-mediated enhanced green fluorescent protein (EGFP) gene in vitro. Fluorescence expression was observed 48 hours later. The rabbit disk degeneration model was established with a microinjection of Fn-f. Ninety-six degenerative disks of 24 rabbits were injected with rAAV2-hTGFß1 (group A), rAAV2-EGFP (group B), or PBS (group C). Immunohistochemical staining for hTGFß1 and fluorescence observation were performed at the 1- and 12-week time points, respectively. 35S-sulfate incorporation assay and Western blot analysis were used to measure the synthesis of proteoglycan and collagen type II at 4-, 8-, and 12-week time points. RESULTS: The dedifferentiated NP cells exhibited intensive fluorescence expression in vitro, with a transfection rate of 90%. In vivo, disks in group A showed enhanced positive hTGFß1 immunostaining at the 1-week time point. At the 4-, 8-, and 12-week time points, disks in group A exhibited significantly increased proteoglycan and collagen type II synthesis compared with the other 2 groups (P<0.01). Abundant green fluorescence was observed in the disks in group B at the 12-week time point. CONCLUSIONS: Early degenerative NP cells are susceptible to AAV-mediated gene transfer in vitro and in vivo. The rapid and prolonged target protein expressions and increased matrix synthesis indicated that AAV-mediated therapeutic gene transfer can be a promising form of treatment for disk regeneration in vivo.

[A novel rabbit disc degeneration model induced by fibronectin fragment].

OBJECTIVE: To establish a novel and useful rabbit model of lumbar disc degeneration using microinjection of fibronectin fragment (Fn-f). METHODS: Thirty-two New Zealand white rabbits underwent injection of N-terminal 30 kDa Fn-f (experimental group) or phosphate buffered saline (PBS) (control group) into the central region of L1-2, L2-3, L3-4, L4-5 discs using a 32-gauge microsyringe. Two rabbits (blank group) with no treatments were sacrificed to examine the proteoglycan synthesis of neucleus pulposus (NP) using (35)S-sulfate incorporation assay. At the 4-, 8-, 12-, and 16-week time points, the discs were examined histologically, radiographically, and with proteoglycan synthesis. RESULTS: Histology demonstrated a progressive loss of the cell numbers in NP and architecture destruction in NP and anulus fibrosus (AF) in Fn-f-injected discs over the 16-week study period. The NP regions in Fn-f-injected discs shrinked distinctly after the 4-week time point, and were not discernible with the inner AF by the 16-week time point. Protoglycan synthesis in Fn-f-injected discs decreased progressively (F = 263.241, P = 0.000). At each time point, the Fn-f-injected discs showed significantly decreased proteoglycan synthesis compared with controls (t = -27.010 - -2.833, P < 0.05). The DHI% of the Fn-f-injected discs at the 4-, 8-, 12-, and 16-week time points were 96.5% ± 1.7%, 85.6% ± 3.8%, 77.2% ± 3.5% and 65.5% ± 5.6%, respectively. Comparing with the DHI% of PBS-injected discs (97.4% ± 1.2%), the Fn-f-injected discs exihibited no significant differences in disc heights at the 4-week time point (P > 0.05), but significant decreases in disc heights at the 8-, 12-, and 16-week time points (t = -21.225 - -10.795, P < 0.01). Apparent anterior osteophytes formed at the 12-week time point and enlarged remarkablely by the 16-week time point in the experimental spines. CONCLUSIONS: Fn-f can induce a progressively degenerative process in rabbit discs which is ethical, cost-effective, reproducible, and consistent with the spontaneous degeneration in human. And it seem to be a novel and useful model for the study of disc degeneration at the molecular level.

A novel rabbit disc degeneration model induced by fibronectin fragment.

OBJECTIVE: To establish a novel and useful rabbit model of lumbar disc degeneration using microinjection of a fibronectin fragment. METHODS: Thirty-two rabbits underwent injection of N-terminal 30 kDa fibronectin fragment (Fn-f) (Group A, n=12; Group B, n=4) or phosphate buffered saline (PBS) (Group C, n=12; Group D, n=4) into the lumbar discs using a 32-gauge microsyringe. Two rabbits (Group E) with no treatment were sacrificed to examine the proteoglycan synthesis of neucleus pulposus (NP) using (35)S-sulfate incorporation assay. At the 4-, 8-, 12-, and 16-week time points, the discs were examined histologically, radiographically and with proteoglycan synthesis. RESULTS: (1) Histology demonstrated a progressive loss of cell numbers in NP and architecture disorganization in NP and annulus fibrosus (AF) over the study period. (2) Radiology: comparing with the PBS-injected discs, the Fn-f-injected discs exhibited no significant differences in disc heights at the 4-week time point, but significant decreases in disc heights at the 8-, 12-, and 16-week time points (P<0.01). Apparent anterior osteophytes formed at the 12-week time point and enlarged remarkably by the 16-week time point in the Fn-f-injected spines. (3) Protoglycan synthesis in the Fn-f-injected discs decreased progressively (P<0.01). At each time point, the Fn-f-injected discs showed significantly decreased proteoglycan synthesis compared with controls (P<0.05 or P<0.01). CONCLUSIONS: Fn-f induced a progressively degenerative process in rabbit discs, which was consistent with the spontaneous degeneration in human. Fn-f induced degeneration seemed to be a novel and useful model for the study of disc degeneration at the molecular level.

Three-dimensional computed tomography imaging increases the reliability of classification systems for tibial plateau fractures.

OBJECTIVE: The purpose of this study was to determine whether three-dimensional reconstructed computed tomography (CT) images can improve intra-observer and inter-observer reliability for classification systems of tibial plateau fractures compared to plain radiographs and two-dimensional CT images. METHODS: Twenty-one tibial plateau fractures were classified independently by four attending orthopaedic trauma surgeons using the AO/ASIF and Schatzker classification systems. First, a combination of plain radiographs and two-dimensional (2D) CT images were evaluated. Second, 4 weeks later, plain radiographs and three-dimensional (3D) CT images were assessed. Then, 4 weeks later, these two rounds of evaluation were repeated. The intra-observer and inter-observer reliability were assessed using kappa statistics. RESULTS: Three-dimensional CT images can improve the inter-observer and intra-observer reliability regarding both AO/ASIF and Schatzker classification systems of tibial plateau fractures compared to 2D CT images. The degree of agreement of the inter-observer and intra-observer reliability among four surgeons increased from 'substantial' to 'almost perfect'. CONCLUSION: Three-dimensional CT is a more reliable radiographic modality than 2D CT in evaluation of fracture patterns in tibial plateau fractures. This finding seems to show that more sophisticated imaging techniques can improve the reliability of fracture classification systems.