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In recent years, mesenchymal cell-based products have been developed to improve surgical therapies aimed at repairing human tissues. In this context, the tooth has recently emerged as a valuable source of stem/progenitor cells for regenerating orofacial tissues, with easy access to pulp tissue and high differentiation potential of dental pulp mesenchymal cells. International guidelines now recommend the use of standardized procedures for cell isolation, storage and expansion in culture to ensure optimal reproducibility, efficacy and safety when cells are used for clinical application. However, most dental pulp cell-based medicinal products manufacturing procedures may not be fully satisfactory since they could alter the cells biological properties and the quality of derived products. Cell isolation, enrichment and cryopreservation procedures combined to long-term expansion in culture media containing xeno- and allogeneic components are known to affect cell phenotype, viability, proliferation and differentiation capacities. This article focuses on current manufacturing strategies of dental pulp cell-based medicinal products and proposes a new protocol to improve efficiency, reproducibility and safety of these strategies.

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Mesenchymal stem cells (MSCs) represent alternative candidates to chondrocytes for cartilage engineering. However, it remains difficult to identify the ideal source of MSCs for cartilage repair since conditions supporting chondrogenic induction are diverse among published works. In this study, we characterized and evaluated the chondrogenic potential of MSCs from bone marrow (BM), Wharton’s jelly (WJ), dental pulp (DP), and adipose tissue (AT) isolated and cultivated under serum-free conditions. BM-, WJ-, DP-, and AT-MSCs did not differ in terms of viability, clonogenicity, and proliferation. By an extensive polychromatic flow cytometry analysis, we found notable differences in markers of the osteochondrogenic lineage between the 4 MSC sources. We then evaluated their chondrogenic potential in a micromass culture model, and only BM-MSCs showed chondrogenic conversion. This chondrogenic differentiation was specifically ascertained by the production of procollagen IIB, the only type II collagen isoform synthesized by well-differentiated chondrocytes. As a pilot study toward cartilage engineering, we encapsulated BM-MSCs in hydrogel and developed an original method to evaluate their chondrogenic conversion by flow cytometry analysis, after release of the cells from the hydrogel. This allowed the simultaneous quantification of procollagen IIB and α10, a subunit of a type II collagen receptor crucial for proper cartilage development. This work represents the first comparison of detailed immunophenotypic analysis and chondrogenic differentiation potential of human BM-, WJ-, DP-, and AT-MSCs performed under the same serum-free conditions, from their isolation to their induction. Our study, achieved in conditions compliant with clinical applications, highlights that BM-MSCs are good candidates for cartilage engineering.

Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31− cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31− DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤ 1%). CD56+, CD146+, CD271+, and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+, and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+, and Stro-1+ cells, and MSCA-1 by 15–30% of CD56+, CD146+, CD271+, and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+, and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products.