We isolated PDL stromal cells from healthy premolars extracted for orthodontic purposes and confirmed that these cells possess a high survival rate, high proliferation rate, and multipotency. In addition, these cells were induced with 5-Aza to form muscle-like structures in vitro.
According to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, MSCs must possess the following minimal criteria.16
First, MSCs must adhere to plastic when maintained in standard culture conditions with tissue culture dishes. Second, MSCs must express the markers CD105, CD73, and CD90, as measured by flow cytometry, but should not express CD45, CD34, CD14, CD11b, CD79a, CD19, or HLA class II. Third, the cells must be capable of differentiating into osteoblasts, adipocytes, and chondrocytes under standard in vitro
differentiation conditions. Our results showed that PDL stromal cells from adult premolars displayed the characteristics of MSCs. During orthodontic treatment, multiple premolars are frequently extracted and discarded as medical waste. It is noteworthy that extracted premolars are a good source of postnatal stem cells, which can be applied in various clinical situations.
Previous studies have reported that stem cells obtained from the outgrowth method may be inferior in quality when compared to the cells from the enzyme digestion method with respect to multipotency and effectiveness of differentiation.6
However, from the clinician's point of view, the enzyme digestion method is more time consuming and more expensive due to the use of multiple enzymes. In addition, the use of enzymes derived from different species may have cross-reaction issues further limiting clinical applications in humans.7
Therefore, we used the outgrowth method following standard protocols. Our results suggest that PDL stromal cells derived from this method have similar proliferation rates as MSCs derived from other tissues. Moreover, the multipotency to differentiate into osteoblast, adipocyte and chondroblast lineages is similar to that previously reported for enzyme-digested PDL stem cells.14
Unlike the substantial number of hard tissue-regeneration strategies based on the application of PDL cells, the potential for skeletal muscle regeneration using PDL stem cells has been scarcely reported.14
Bone and muscle differentiation is modulated in a complementary manner; muscle progenitor cells frequently differentiate into osteoblasts by the suppression of muscle differentiation factors.17
MSCs possess the potential for osteocyte differentiation; therefore, myogenesis of DP stem cells is ineffective in standard myogenic medium.12
However, the treatment of stem cells with 5-Aza prior to myogenic induction has been reported to cause successful differentiation of mouse DP stem cells into myotubes.12
Pretreatment with 5-Aza results in demethylation of the DNA of genes encoding muscle-specific transcriptional factors, which under other circumstances is inhibited by the methylation of the transcriptional regulatory region.12
In a manner similar to that for DP stem cells, we were able to induce the differentiation of human PDL stromal cells into myotubes by pretreatment of the cells with 5-Aza, but not in its absence.
Many clinical orthodontists are aware of the importance of soft tissues in oromaxillofacial regeneration. Masticatory muscle volume and function are often critically important for diagnosis, treatment efficiency, positive outcomes, and long-term prognosis. The results of this study indicate that PDL stromal cells isolated from orthodontically extracted premolars may be a good source of postnatal stem cells for oromaxillofacial regeneration of hard tissues, as well as soft tissues, including the facial muscles.