Basic Study
Copyright ©The Author(s) 2025. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. May 26, 2025; 17(5): 104116
Published online May 26, 2025. doi: 10.4252/wjsc.v17.i5.104116
Optimal good manufacturing practice-compliant production of dental follicle stem cell sheet and its application in Sprague-Dawley rat periodontitis
Jia-Lu Yu, Chao Yang, Li Liu, An Lin, Shu-Juan Guo, Wei-Dong Tian
Jia-Lu Yu, An Lin, State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, Sichuan Province, China
Chao Yang, Department of Product Development, Chengdu Shiliankangjian Biotechnology Co., Ltd, Chengdu 610041, Sichuan Province, China
Li Liu, Engineering Research Center of Oral Translational Medicine, National Clinical Research Center for Oral Diseases, Departments of 5 Periodontics and 6 Oral and Maxillofacial Surgery, Sichuan University, Chengdu 610041, Sichuan Province, China
Shu-Juan Guo, Department of Periodontics, West China School of Stomatology, Sichuan University, Chengdu 610041, Sichuan Province, China
Wei-Dong Tian, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Engineering Research Center of Oral Translational Medicine, National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, Sichuan Province, China
Co-corresponding authors: Shu-Juan Guo and Wei-Dong Tian.
Author contributions: Yu JL, Yang C, Guo SJ, and Tian WD contributed to the conceptualization and methodology; Yu JL, Liu L, and Lin A were involved in the investigation and formal analysis; Yu JL wrote original draft; Yang C and Guo SJ contributed to the revision of this manuscript; Guo SJ and Tian WD provided daily supervision, project administration, and funding acquisition, they contributed equally to this manuscript as co-corresponding authors.
Supported by National Key Research and Development Program of China, No. 2021YFA1100600 and No. 2022YFA1104400.
Institutional review board statement: The study was reviewed and approved by the Ethics Committee of the West China School of Stomatology, Sichuan University, (Approval No. WCHSIRB-D-2024-237).
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the West China School of Stomatology, Sichuan University, IACUC protocol number: Protocol No. WCHSIRB-D-2024-368.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Data sharing statement: The data supporting the findings of this study are available from the corresponding author upon reasonable request.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Wei-Dong Tian, State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Engineering Research Center of Oral Translational Medicine, National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3, Renmin South Road, Wuhou District, Chengdu 610041, Sichuan Province, China. drtwd@sina.com
Received: December 11, 2024
Revised: February 27, 2025
Accepted: April 18, 2025
Published online: May 26, 2025
Processing time: 167 Days and 5.8 Hours
Abstract
BACKGROUND

Dental follicle stem cell (DFSC) sheets demonstrate strong extracellular secretion capabilities and efficacy in periodontal regeneration. However, existing methods for producing DFSC sheets lack a comprehensive discussion on the most efficient and cost-effective approaches at the good manufacturing practice (GMP) level.

AIM

To investigate the culture condition of GMP-compliant DFSC sheets and to compare the properties of DFSC sheets and cell suspensions.

METHODS

This study explored the optimal conditions for culturing GMP-compliant DFSC sheets, focusing on four key factors: Cell passage, cell concentration, L-ascorbic acid content, and culture duration. We evaluated the characteristics of the cell sheets under varying culture conditions, including cell viability, cell count, appearance, osteogenesis, chondrogenesis, odontogenesis, aging, relative telomere length, and extracellular matrix secretion. A comparison was also made between the periodontal regeneration, osteogenesis, and paracrine capacity of cell sheets cultured under optimal conditions and those of the cell suspensions.

RESULTS

The GMP-compliant DFSC sheets cultured from passage 4 cells exhibited the highest viability (≥ 99%, P < 0.05) and optimal osteogenic differentiation capacity (optical density ≥ 0.126, P < 0.05). When cultured for 10 days, DFSC sheets demonstrated maximal expression of osteogenic, chondrogenic and periostin genes [alkaline phosphatase, Runt-related transcription factor 2, collagen type I, osteopontin, cartilage associated protein, and PERIOSTN (P < 0.001); osteocalcin (P < 0.01)]. Concurrently, they showed the lowest senescent cell count (P < 0.01) with no progression to late-stage senescence. At a seeding density of 2500 cells/cm2, GMP-compliant DFSC sheets achieved better osteogenic differentiation (P < 0.01) and maximal osteogenic, chondrogenic and periostin gene expression (P < 0.001), coupled with the highest hydroxyproline secretion (P < 0.001) and moderate sulfated glycosaminoglycan production. No statistically significant difference in senescent cell count was observed compared to DFSC sheets at a seeding density of 5000 cells/cm2. Supplementation with 25 μg/mL L-ascorbic acid significantly enhanced osteogenic gene expression (P < 0.001) and elevated hydroxyproline (P < 0.01) and sulfated glycosaminoglycan secretion to high ranges. Compared with the cell suspension, the cell sheet demonstrated improved osteogenic, paracrine, and periodontal regenerative capacities in Sprague-Dawley rats. The optimized DFSC sheets demonstrated significantly higher levels of vascular endothelial growth factor and angiopoietin-1 (P < 0.001) compared to DFSC suspensions, along with enhanced osteogenic induction outcomes (optical density = 0.1333 ± 0.01270 vs 0.1007 ± 0.0005774 in suspensions, P < 0.05). Following implantation into the rat periodontal defect model, micro-computed tomography analysis revealed superior bone regeneration metrics in the cell sheet group compared to both the cell suspension group and control group (percent bone volume, trabecular thickness, trabecular number), while trabecular spacing exhibited an inverse pattern.

CONCLUSION

Optimized DFSC sheets cultured under the identified conditions outperform DFSC suspensions. This study contributes to the industrial-scale production of DFSC sheets and establishes a foundation for cell therapy applications.

Keywords: Periodontitis; Dental follicle stem cells; Cell sheet; Periodontal tissue regeneration; Mesenchymal stem cells; Vitamin C; Cell concentration; L-ascorbic acid

Core Tip: The existing culture method for dental follicle stem cell (DFSC) sheets is crude and not uniform. For clinical application, it is necessary to produce good manufacturing practice compliant DFSC sheets and determine a uniform culture method that can maintain the best performance of DFSC sheets and consume the least. In this study, the culture conditions of the good manufacturing practice-compliant DFSC sheet were optimized, focusing on four key factors: Cell passage, cell concentration, L-ascorbic acid content, and culture duration. Compared with the DFSC suspension, the cell sheet demonstrated superior capacities. These findings offer valuable insights and guidance for the clinical application of cell sheets.