Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers

Abstract

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51⁺/CD140α⁺, 0.8% were CD271⁺, and 2.4% were STRO-1⁺/CD146⁺. Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271⁺ DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

Introduction

Mesenchymal stem cell (MSC)-mediated therapy has been shown to produce promising clinical outcomes in regenerating tissue defects for regenerative medicine and periodontal therapy.^1,2 Human MSCs are multipotent adult progenitor cells with the capability to self-renew and the potential to differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes.^3,4 Additional characteristics of MSCs, including their ease of isolation and lack of immunogenicity, make them unique and indispensable tools in tissue engineering and regenerative therapy.^5,6 While bone marrow is the most widely recognized source of MSCs (bone marrow-derived mesenchymal stem cells (BMSCs)), additional sources for MSCs including craniofacial tissues have been identified.^7,8,9 In particular, periodontal ligament (PDL) tissue collected from extracted adult and primary teeth is an easily accessible source of MSCs and such MSCs isolated from the PDL were found to have the ability to regenerate bone, cementum, and PDL upon in vivo transplantation.^10,11,12 As such, the PDL has been identified as a viable and promising source for MSCs in promoting regenerative therapy, especially for craniofacial defects such as periodontal disease.^8,11,12

The PDL is a dynamic and specialized connective tissue derived from the dental follicle that originates from neural crest cells.^13,14 PDL tissues contain a heterogeneous population of cells, including fibroblasts, epithelial cells, endothelial cells, cementoblasts, osteoblasts, and neural cells.¹⁵ Embedded between the cementum and the inner wall of the alveolar bone socket, the PDL’s primary functions are to anchor the teeth to the alveolar bone and to provide them with protection against mechanical loads generated by mastication.¹⁶ In addition to mechanical support, the PDL has many critical biological functions including providing tooth nutrition and regenerating periodontal tissues damaged by inflammatory periodontal disease or mechanical trauma.¹⁶

The role of the PDL is especially important in repair after periodontal disease, which can have acute, chronic, or systemic manifestations, ultimately leading to destruction of periodontal tissue, progressive alveolar bone loss, and eventual tooth loss.^{17,18,19,20,21} This periodontal regeneration is challenging due to the complexity of the PDL attachment apparatus requiring finely orchestrated formation of new cementum, bone, and PDL fibers followed by the insertion of these fibers into the bone and cementum.^51,52,53,54 In our study, a high proportion (24%) of PDLCs were positive for CD51 and CD140α. This large yield of CD51⁺/CD140α⁺ DMSCs from the PDL is consistent with high Nestin expression in the majority of adult neural crest stem cells.^55,56 In addition, these isolated CD51⁺/CD140α⁺ DMSCs were successfully induced to undergo differentiation into osteogenic and chondrogenic lineages. Quantification of ALP activity and ARS and expression of osteogenic marker genes exhibited comparably significant osteogenic and chondrogenic potential for CD51⁺/CD140α⁺ DMSCs as STRO-1⁺/CD146⁺ DMSCs.

Our findings not only offer recommendations for isolating MSCs from PDL, but also provide future directions for clinical applications of DMSCs in dentistry and medicine, including periodontal therapy. Periodontal disease is a major cause of tooth loss and a substantial public health concern.¹⁹ Caused by precipitating factors such as microorganisms and their byproducts, periodontitis is initiated by an inflammatory process that leads to the dissolution of tissue components.^17,20 The selective isolation of DMSCs from the PDL may offer the possibility of improvements in regenerating the periodontal apparatus that is destroyed by periodontal disease. Additional clinical applications of DMSCs from PDL in dentistry include periodontal defect repair, PDL development for titanium dental implants, and tooth root repair.^12,27,57,58

In conclusion, our findings demonstrated the successful isolation of distinct subpopulations of DMSCs from human PDL with the use of CD51/CD140α, CD271, and STRO-1/CD146 surface markers and demonstrated their capacity to undergo differentiation into osteogenic and chondrogenic lineages. Each marker yielded a different quantity of isolated mesenchymal progenitor cells with varying magnitude of multi-lineage differentiation potential. As CD51/CD140α produced isolation of significantly higher proportion of PDLCs than the other two cell surface marker combinations, CD51/CD140α may be a sufficient marker combination to use with FACS analysis to obtain highly multipotent MSCs from the PDL. Further studies are needed to validate whether these isolated cells may differentiate into functionally different lineages in vivo.