Extracellular matrix synthesis and ultrastructural changes of degenerative disc cells transfected by Ad/CMV-hTGF-beta 1 / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To determine whether the synthesis of proteoglycan, collagen and associated ultrastructure are related to the adenovirus-mediated gene transferred to adult degenerative cells.</p><p><b>METHODS</b>Adenovirus/cytomegalovirus human transforming growth factor-beta 1 (Ad/CMV-hTGF-beta 1) was used to transfect degenerative cells. Antonopulos method, Miamine method and transmission electron microscopy were conducted to study the synthesis of proteoglycan, collagen, and ultrastructure, respectively. Cell cultures were established from the nucleus pulpous and annulus fibrosus tissues, which were taken from surgery.</p><p><b>RESULTS</b>Nucleus pulpous and annulus fibrosus cells were efficiently transduced by the adenoviral vector carrying hTGF-beta 1 gene. The synthesis of proteoglycan and collagen increased compared with the control group (P < 0.05). The metabolism of cells was slightly improved. No significant toxic effects were found.</p><p><b>CONCLUSIONS</b>Expression of hTGF-beta 1 gene is efficient to accelerates proteoglycan synthesis and thus accelerates the improvement of collagen. The function and structure of degenerative cells are improved. Ad/CMV-hTGF-beta 1 may be suitable for treating disc degeneration.</p>

Construction of recombinant adenoviral vector Ad-CMV-hTGFbeta1 for reversion of intervertebral disc degeneration by gene transfer / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To provide a highly efficient adenoviral vector Ad-CMV-hTGFbeta1 for the study of gene therapy for reversion of the intervertebral disc degeneration.</p><p><b>METHODS</b>A newly developed recombinant adenoviral vector construction system was used in the study. The cDNA of hTGFbeta1 was first subcloned into a shuttle plasmid pShuttle-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease PmeI, and subsequently transformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected into 293 cells. Recombinant adenoviruses were generated within 2 weeks.</p><p><b>RESULTS</b>The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The expression of hTGFbeta1 was verified by immunohistochemical staining.</p><p><b>CONCLUSIONS</b>The successful generation of the adenoviral vector Ad-CMV-hTGFbeta1 and the confirmation of the interest gene expression make it possible for the experimental study of the reversion of the intervertebral disc degeneration by gene therapy.</p>

Comparison of the fatigue properties of bone cement specimens prepared with different monomer storing temperatures and different polymerization ambient temperatures / 中华骨科杂志

Objective To investigate the fatigue properties of bone cement specimens prepared with different monomer storing temperatures and polymerization environment temperatures; to find out the combination of the storing temperature of bone cement monomer and the mixing temperature of bone cement. Methods Bone cement monomer was stored under 21 ℃ or 4 ℃ environment. They were mixed with the powder under partial vacuum at room temperature of 21 ℃ for 1 minute, followed by 3 minutes' rest. The cement was then injected into metal molds with different temperatures of 21 ℃ or 50 ℃. Finally, the polymerized cement specimens were removed from the molds for mechanical tests. The bone cement specimens were divided into four groups. Group 1(control group): monomer temperature 21 ℃, metal mold temperature 21 ℃. Group 2: monomer 21 ℃, mold 50 ℃. Group 3: monomer 4 ℃, mold 21 ℃. Group 4: monomer 4 ℃, mold 50 ℃. Each group consisted of 21 specimens. All specimens were soaked in 37 ℃ water bath, and were tested with MTS mechanical test machine. Completely reversed tension-compression cycling loading was applied. Testing strength exerted was 15 MPa and the frequency was 2 Hz. This strength was approximately 3.5 times of the force that cement received in vivo. The cycles to failure was recorded. Specimens that failed in less than 5000 cycles were excluded. Comparisons of cycles to failure among different groups were performed using independent sample t tests. Results The cycles to failure of the control group(group 1) were lower compared with those of group 2, 3 and 4. The differences were all statistically significant(P≤0.001). Conclusion 1) The combination of monomer temperature of 21 ℃ and metal mold (polymerization environment) temperature of 21 ℃ produced the worst bone cement specimens in terms of their fatigue property. 2) The fatigue property was obviously improved when the monomer and the mold temperatures differed by 20 ℃ to 30 ℃.