Breaking Barriers: Evaluating Challenges in Advancing Periodontal Ligament Cell-Derived Organoids

Abstract

The objective of this review was to critically evaluate the available literature on the development of periodontal ligament organoids. Articles concerning periodontal ligament organoids were considered eligible. References were selected in a two-phased process. Electronic databases PubMed and Scopus were screened up to June 2024, yielding 1101 studies. After removing duplicates, titles, and abstracts were screened, resulting in 44 articles being included in this review. A detailed analysis of the included articles was organized into four categories: (1) the cell lineages used, including the simultaneous use of two or more cell types, (2) the extracellular matrix composition, (3) the organoid preparation methods, and (4) the characterization techniques employed. The main findings show that collagen combined with biodegradable polymers-such as poly(caprolactone), poly(glycolic acid), and poly(lactic acid)-is the most used material. Periodontal ligament cells and periodontal fibroblasts were the most used cell types, due to their role in extracellular matrix remodeling. The most frequent analyses performed included alkaline phosphatase, extracellular matrix mineralization, and gene expression, providing insights into differentiation and periodontal regeneration. Cementogenic differentiation was the most studied, followed by osteogenic, chondrogenic, adipogenic, and epithelial differentiation. However, challenges remain, including methodological inconsistencies and the need for scaffold optimization. Future research should focus on standardizing protocols, improving biomaterials, and integrating bioprinting techniques to improve clinical translation.

Keywords: organoids; periodontal ligament cells; periodontal regeneration; three-dimensional cell culture.

Figure 1

Flowchart of the literature search and selection of the included studies. References were selected in a two-phase process. Electronic databases (PubMed and SCOPUS) were searched up to June 2024.

Figure 2

Venn diagram concerning the cell types, origin, and groups employed for organoid construction. Cell lineages employed for organoid construction included alveolar bone cells (n = 1), alveolar bone stem cells (n = 1), dental pulp stem cells (n = 1), dental epithelium cells (n = 1), dental epithelium stem cells (n = 1), cementoblasts (n = 2), dental follicle cells (n = 2), immortalized periodontal ligament cells (n = 2), gingival fibroblasts (n = 5), periodontal ligament stem cells (n = 6), and periodontal ligament cells or periodontal ligament fibroblast (n = 34). Most included studies used one cell type (n = 36), followed by studies using two types (n = 8) and three types (n = 3). Human cells (n = 42) were used in most included studies, followed by cells from mice (n = 3) and from porcine (n = 1).

Figure 3

Venn diagram demonstrating the extracellular matrix component used for organoid fabrication.

Figure 4

Methods used for organoid preparation. (A) A solid scaffold is made by extracellular matrix composition solution, and the cell suspension is seeded on the top of the scaffold. Introduce the cell suspension inside the extracellular matrix (ECM) composition solution and mix to development the organoid. (B) Introduce the cell suspension inside the extracellular matrix (ECM) composition solution and mix to development the organoid.

Figure 5

Venn diagram demonstrating findings concerning (A) cellular viability, proliferation, adhesion, migration, orientation, and (B) differentiation. (C) Organoid surface and mechanical characteristics were also evaluated. Legend: (A) CCK-8 assay—Cell Counting Kit-8; EdU incorporation—thymidine analog (5-ethynyl-2′-deoxyuridine) incorporation; FDA-P1 method—fluorescein diacetate; LDH assay—lactate dehydrogenase assay; MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); MTT—(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); TSO SPS microscope—Triple Staining Observation with Structured Pattern Scanning; WST-1—Water-Soluble Tetrazolium Salt-1. (B) ELISA—enzyme-linked immunosorbent assay; RT-PCR—Real-Time Polymerase Chain Reaction; RNA-seq—RNA sequencing.