Is There a Noninvasive Source of MSCs Isolated with GMP Methods with Better Osteogenic Potential?

Carla C G Pinheiro, Alessander Leyendecker Junior, Daniela Y S Tanikawa, José Ricardo Muniz Ferreira, Reza Jarrahy, Daniela F Bueno, Carla C G Pinheiro, Alessander Leyendecker Junior, Daniela Y S Tanikawa, José Ricardo Muniz Ferreira, Reza Jarrahy, Daniela F Bueno

Abstract

Background: A new trend in the treatment for alveolar clefts in patients with cleft lip and palate involves the use of bone tissue engineering strategies to reduce or eliminate the morbidity associated with autologous bone grafting. The use of mesenchymal stem cells-autologous cells obtained from tissues such as bone marrow and fat-combined with various biomaterials has been proposed as a viable option for use in cleft patients. However, invasive procedures are necessary to obtain the mesenchymal stem cells from these two sources. To eliminate donor site morbidity, noninvasive stem cell sources such as the umbilical cord, orbicularis oris muscle, and deciduous dental pulp have been studied for use in alveolar cleft bone tissue engineering. In this study, we evaluate the osteogenic potential of these various stem cell types.

Methods: Ten cellular strains obtained from each different source (umbilical cord, orbicularis oris muscle, or deciduous dental pulp) were induced to osteogenic differentiation in vitro, and the bone matrix deposition of each primary culture was quantified. To evaluate whether greater osteogenic potential of the established mesenchymal stem cell strains was associated with an increase in the expression profile of neural crest genes, real-time qPCR was performed on the following genes: SRY-box 9, SRY-box 10, nerve growth factor receptor, transcription factor AP-2 alpha, and paired box 3.

Results: The mesenchymal stem cells obtained from deciduous dental pulp and orbicularis oris muscle demonstrated increased osteogenic potential with significantly more extracellular bone matrix deposition when compared to primary cultures obtained from the umbilical cord after twenty-one days in culture (p = 0.007 and p = 0.005, respectively). The paired box 3 gene was more highly expressed in the MSCs obtained from deciduous dental pulp and orbicularis oris muscle than in those obtained from the umbilical cord.

Conclusion: These results suggest that deciduous dental pulp and orbicularis oris muscle stem cells demonstrate superior osteogenic differentiation potential relative to umbilical cord-derived stem cells and that this increased potential is related to their neural crest origins. Based on these observations, and the distinct translational advantage of incorporating stem cells from noninvasive tissue sources into tissue engineering protocols, greater study of these specific cell lines in the setting of alveolar cleft repair is indicated.

Conflict of interest statement

The authors declare that they have no competing interests.

Copyright © 2019 Carla C. G. Pinheiro et al.

Figures

Figure 1
Figure 1
Morphology of adherent cells after isolation from corresponding tissue (sources): (a) orbicular oris muscle-derived stem cell (OOMDSC); (b) dental pulp stem cell (DPSC); (c) umbilical cord mesenchymal stem cell (UC-MSC). Similarity in the fibroblastoid morphology among the three different strains is observed.
Figure 2
Figure 2
Multilineage differentiation in vitro. Row A: OOMDSC; row B: DPSC; and row C: UC-MSC. (a) The control group of undifferentiated strains. (b) Adipogenic differentiation after eighteen days of induction and staining with oil red; white arrows show the fat vesicles. (c) Chondrogenic differentiation after 3 weeks of induction, stained with alcian blue; white arrows show the extracellular matrix formation—mucopolysaccharides. (d) Osteogenic differentiation after 3 weeks of OOMDSC induction, stained with alizarin red S; white arrows show the extracellular matrix deposition.
Figure 3
Figure 3
Quantitative measurement of the extracellular bone matrix stained with alizarin red S. Graphical representation of the measurement of the extracellular bone matrix deposited during osteogenic differentiation induction at 0, 3, 7, 14, and 21 days, showing the beginning of the deposition of extracellular matrix after 7 days of induction in vitro with increases on days 14 and 21.
Figure 4
Figure 4
Neural crest expression in MSCs: relative expression of 5 neural crest genes in undifferentiated DPSC, OOMDSC, and UC-MSC strains. This experiment was repeated with three replicates for each sample (n = 10). The data are presented as the mean +/− (∗ represents the outlier data).

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