Noggin and Chordin knockdown in distraction osteogenesis

Introduction: Numerous reports have shown that recombinant BMPs have positive effects in several conditions associated with poor bone formation. We hypothesize that by inhibiting BMP antagonists Noggin and Chordin using RNA interference we may upregulate endogenous BMP expression and enhance osteogenesis. Methods: MC3T3-E1 cells were transduced with lentiviruses expressing various shRNAs targeting the mouse Noggin (5 shRNAs) and Chordin (3 shRNAs) genes and 1 negative control. At various time points after infection, levels of RNA expression for Noggin and Chordin were monitored through RT-PCR. Western blotting were performed on the cell extracts and culture media to verify the expression and secretion of Noggin and Chordin proteins. Cell extracts were also analyzed on day 2 and 4 after transduction for alkaline phosphastase activity which is a marker of osteogenic differentiation. DO was performed on the right tibia of 54 wild-type mice using a miniature Illizarov distraction device. Animals were randomized into two major groups according to the time of sacrifice: end of distraction (day 17) and mid-consolidation (day 34). Each major group was sub-divided in 3 subgroups representing the type of injection that was administered at the site of distraction on day 8. For each time point, 9 mice were injected with PBS, 9 with a lentivirus plasmid containing a Non-Target (NT) shRNA and 9 with lentivirus plasmids containing shRNAs targeting Noggin-Chordin. To evaluate the success of the infection a GFP marker was added to lentivirus plasmid targeting Noggin-Chordin. For each subgroup 6 collected samples were studied using Faxitron X-ray, μCT and immunohistochemistry and 3 were studied using Rt-PCR. Results: On non-tranduced MC3T3-E1 cells, the levels of RNA expression of Noggin and Chordin was at its highest level on Day 7. At this timepoint, qRT-PCR analysis showed that the shRNAs were effective in knocking down Noggin and Chordin endogenous mRNA levels down to 10% and 17%, respectively, by the most potent of shRNAs tested compared to control. Western Blot analysis also corroborates that the respective shRNAs were effective in knocking down the Noggin and Chordin proteins. Specific activity of alkaline phosphatase was increased in MC3T3-E1 cells stably expressing Noggin and Chordin shRNA.Concerning our in vivo work, all the surgeries and distraction were successful except for the death of 2 animals secondary to post-operative complications. However, at the time of the injection the majority of the injection content was extravasating out of the injection site. Once the samples collected, the Faxitron X-ray and μCT studies did not show a significant difference of bone volume or in the ratio of bone volume/tissue volume at the distraction site in between the 3 injection subgroups at both time points. The Rt-PCR studies did not show a significant difference in inhibiting Noggin or Chordin mRNA levels in between the 3 subgroups at both time points. Finally, the immunohistochemistry studies were stopped after the GFP marker was found in all the three subgroups. Conclusion: In our in vitro study, western blot analysis, qRT-PCR and ALP assay were consistent with a successful knockdown of Noggin and Chordin. During our in vivo study, the main issues faced during this project were technical problems during the injection. Unfortunately, our study did not show any significant difference in bone formation after using injection of lentivirus plasmid with shRNA targetting BMPs antagonists Noggin and Chordin.