Re: Response to: Bone marrow mesenchymal stem cells enhance bone formation in orthodontically expanded maxillae in rats. Abdullah Ekizer, Mehmet Emir Yalvac, Tancan Uysal, Mehmet Fatih Sonmez, Fikrettin Sahin. The Angle Orthodontist. Online Early

To: Editor, The Angle Orthodontist

Thank you for the comments made on our paper. We believe that this study is a novel proof of principle study and it suggests potential use of mesenchymal stem cells (MSCs) after maxillary expansion. Based on this report, we and other groups can work on improving the technique and testing the efficacy of MSCs in reducing relapse rates compared to the other options.

We apologize for the mistake in our reported Results and have made corrections in the text and in Table 2:

“Our histomorphometric analysis revealed that the group injected with MSCs had higher new bone formation than the PBS-injected group (P <.05; 1.06-fold). In correlation with this, we have found that the number of osteoblasts (P <.05; 1.2-fold) and vessels (P <.05; 1.71-fold) was higher in the MSC-treated group. On the other hand, we did not find a significant difference in the number of osteoclasts (P  = .288) between the two groups (Table 2).”

Since this was a proof of principle study, we only showed the effect of MSCs. We are currently performing further studies to improve this technique in terms of dose, delivery volume, use of bio-polymers along with MSCs, and pre-differentiation of MSCs before injection. Once a well-established, clinically-relevant treatment approach is developed, we will be able to assess the efficacy of stem cell treatment compared to the other methods more accurately.

It is not accurate to assume that all cells stained positive with DAPI are apoptotic. DAPI is a nuclear counter stain and it is used to stain whole cells in tissues to determine the presence of the cell nuclei in immunofluorescence imaging. In our images, all of the cells including endogenous and transplanted MSCs were nicely stained with DAPI. Identification of transplanted MSCs was done by determining DAPI⁺ and pkh67⁺ cells. This is a very standard way of showing certain cell populations in vivo using the immunofluorescence technique. DAPI staining can only show the apoptotic cells with compartmentalized nuclei at a high magnification. In this case, we have not detected any abnormal looking cell nuclei in either the pkh67⁺ cells or endogenous cells during histological analysis. If there was apoptosis or necrosis in the areas we showed in this article, there would be massive inflammation and impaired regeneration which was not detected in this study. There are certainly better ways of detecting apoptotic cells in vivo such as TUNEL assay which could be employed if needed as a gold standard.

Regarding the suggestion that we provide further characterization of MSCs used in this study, this could increase the quality of the paper. However, we believe that our flow cytometry data and differentiation assays performed as previously published by our group^1,2 was sufficient for describing MSCs characteristic of transplanted cells in this study.

Overall, this was a novel proof of principle study and the techniques of delivery of MSCs still need to be improved. When the stem cells are injected, they are not floating in the PBS but reside in the tissue where a plethora of cytokines and chemokines are guiding stem cells through their localization and differentiation as well as their paracrine secretion of important growth factors such as FGF2, BMP2.³ The dose of the cells and chemistry of the vehicle can dramatically change the efficacy of the treatment. Our study, being novel, will encourage others groups to perform similar experiments and this will help us to move forward in using MSCs in orthodontic treatment.

The questions outlined are all topics for further studies about stem cell use in orthodontics. We apologize again for the mistyped data in the table that caused some minor confusion. Overall, all issues raised are far from fairly assessing the value of the paper. We think that a further reading will reveal that there is currently a lack of studies regarding the use of stem cells after maxillary expansion and we believe that the elucidations given in this proof of principle paper should be viewed from that perspective.