Optimal good manufacturing practice-compliant production of dental follicle stem cell sheet and its application in Sprague-Dawley rat periodontitis.

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%, < 0.05) and optimal osteogenic differentiation capacity (optical density ≥ 0.126, < 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 ( < 0.001); osteocalcin ( < 0.01)]. Concurrently, they showed the lowest senescent cell count ( < 0.01) with no progression to late-stage senescence. At a seeding density of 2500 cells/cm, GMP-compliant DFSC sheets achieved better osteogenic differentiation ( < 0.01) and maximal osteogenic, chondrogenic and periostin gene expression ( < 0.001), coupled with the highest hydroxyproline secretion ( < 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/cm. Supplementation with 25 μg/mL L-ascorbic acid significantly enhanced osteogenic gene expression ( < 0.001) and elevated hydroxyproline ( < 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 ( < 0.001) compared to DFSC suspensions, along with enhanced osteogenic induction outcomes (optical density = 0.1333 ± 0.01270 0.1007 ± 0.0005774 in suspensions, < 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.