Exploring the impact of culture techniques and patient demographics on the success rate of primary culture of human periodontal ligament stem cells

Abstract

Background/purpose: Periodontal ligament stem cells (PDLSCs) have the potential for regenerating periodontal tissue. The study aims to investigate the impact of demographics (ages, gender, disease) and culture techniques (shipping storage time and culture method) on the success of primary culture.

Materials and methods: PDLSCs were collected from 51 teeth of 26 patients and cultured via outgrowth (OG) and enzymatic digestion (ED) methods. Cells characteristics were confirmed by flow cytometry, MTT, and ARS. The primary culture success rate was evaluated with a serial chi-square test to determine the relationship with culture technique (ED/OG and ≤4 h/prolonged culture) and patient demographics (Young/Old, Female/Male, and Health/Periodontitis).

Results: The overall success rate of Health group (69.7%) was higher than Periodontitis (38.9%). Culturing within 4 h possessed a higher success rate (71.8%) than prolonged group (16.7%) regardless of patient demographics, and using OG method (81.5%) revealed more promising. Subgroup analysis of 39 cases (culture within 4 h) found that the success rate of OG was higher than ED in the Old group (87.5%-25.0%) and in the Periodontitis group (83.3%-25.0%).

Conclusion: Primary culturing of PDLSCs within 4 h and using the outgrowth method led to higher success rates regardless of patient demographics. It can achieve successful PDLSCs culture of older patients or patients with periodontal disease by appropriate culture technique.

Keywords: Cell banking; Enzymatic digestion; Outgrowth; PDLSCs; Primary culture.

Figure 1

Experimental scheme of human periodontal ligament stem cells (PDLSCs) primary culture with Enzymatic digestion (ED) and outgrowth method (OG). (A) Human PDLSCs were primary cultured with Enzymatic digestion and outgrowth methods. 51 teeth, of which 33 were healthy teeth (Health) and 18 were extracted from teeth with periodontitis (Perio). Cell viability, cell differentiation and cell identification were performed for patient matched sample. (B) ED method: root surface was scraped with surgical blade, cut into small pieces, and treated with collagenase. OG method: whole root was explanted without collagenase. CD marker: cluster of differentiation; MTT: MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay; ARS: Alizarin Red S, an anthraquinone dye, to evaluate calcium deposits in differentiated cells.

Figure 2

Microscopic observation in human primary cells of periodontal ligament cells (PDLCs) and gingival fibroblasts (GFs) were cultured with enzymatic digestion or outgrowth culture method. (A) Enzymatic digestion of PDLCs were cultured for 48 h. (B) Outgrowth of PDLCs migrated from the tooth root (“Root” in the upright corner) after 48 h. (C) Outgrowth of PDLCs were shown obviously high proliferation status after 48 h. (D) Outgrowth of GFs from the gingival tissue were cultured for 48 h.

Figure 3

Summarized the success rate in different patient groups and cultural method groups. Success rate in Health and in periodontitis (Perio) periodontal ligament stem cells (PDLSCs) with enzymatic digestion (ED) and outgrowth (OG) primary culture methods. (∗P < 0.05, ∗∗∗P < 0.001).

Figure 4

The results of cell viability and differentiation were according to patient matched data. (A) Periodontal ligament stem cells (PDLSCs) viability of MTT assay was tested on ED and OG primary culture methods in Health and Perio groups. (B) Alizarin red S staining of cell mineralization was tested in healthy PDLSCs and gingival mesenchymal stem cells (GMSCs) at day 2 and day 14. (C) The optical density (O.D.) at 548 nm represents mineral ability in cultured PDLSCs and GMSCs. (∗∗P < 0.01, ∗∗∗P < 0.001).

Figure 5

The isolated periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) were stained with monoclonal antibodies against to CD 34, CD49d, CD90, CD106 and CD166 followed by flow cytometric analysis. Both PDLSCs and GMSCs CD34 are negative; CD90, CD106 and CD166 are positive. GMSCs is higher in CD49d than PDLSCs.