This work aimed to investigate the potential of dental pulp stem cell-derived decellularized extracellular matrix (dECM) for promoting the adhesion and migration of gingival fibroblasts. Our study aimed to comprehensively understand the interactions between the dECM and gingival fibroblasts. The presence of collagen type I and fibronectin in the ECM and dECM derived from DPSCs indicates the composition and characteristics of these matrices. Additionally, the observation of negative intracellular proteins and nuclei within the dECM highlights the successful removal of cellular components during the decellularization process. Proteomic database analysis revealed enrichment of several proteins involved in ECM organization, ECM-receptor interaction, and focal adhesion. These findings shed light on the molecular composition and functional characteristics of the ECM derived from DPSCs.
Understanding how this bioengineered matrix affects cell adhesion and migration is essential for oral tissue regeneration research. GFs were evaluated in comparison to the controls. In our study, we found that collagen type I is abundant in both the ECM and dECM derived from DPSCs. Using collagen type I-coated dishes as a positive control enabled us to evaluate the performance of the dECM. The stress fibers of gingival fibroblasts grown on dECM had a greater degree of organization. In addition, gingival fibroblasts demonstrated a greatly improved capacity for migration and proliferation when grown on dECM. It is likely that gingival fibroblasts grown on dECM exhibit increased mRNA expression of genes involved in the biological functional impact of dECM.
Our previous studies demonstrated that ECM derived from DPSCs expressed ECM proteins in both the ECM and dECM. Decellularization involves removing cellular components while preserving the structural integrity and bioactive molecules of the ECM (8, 9). The successful removal of these intracellular proteins ensures that the dECM is free from cellular remnants, reducing potential immunological reactions and making it suitable for various tissue engineering applications (16). Type I collagen, a fundamental component of the ECM, provides structural support and helps maintain tissue integrity (1). Its presence in both the ECM and dECM indicates that DPSCs can produce this crucial protein, which is essential for tissue structure and function. Fibronectin contributes to the ability of the matrix to support cell behavior, such as adhesion and spreading.
In addition, limitations such as ECM protein loss, incomplete decellularization due to high confluence, variations in chemical concentration and incubation time, ECM disruption before recellularization, and potential cytotoxicity or immunological responses have been identified (17). Enrichment analysis of ECM proteins was conducted using samples from three different donors. A total of 163, 116, and 57 proteins were detected in the respective samples, 33 of which were common (Supplemental Figure 1A). The loss of ECM proteins during the decellularization process did not significantly affect the enrichment of protein‒protein interactions (Supplemental Figure 1B). Furthermore, the expression of ECM proteins in DPSCs is influenced by the conditions of the donor teeth. Compared with immature dental pulp cells, mature dental pulp cells exhibit stronger expression of collagen type I, fibronectin, and tenascin, while vimentin shows consistent expression levels (18). Hence, variations in ECM protein expression among patients may be attributed to genetic differences (19). The concept of patient-specific ECM signatures underscores the uniqueness of each individual's ECM composition and warrants further investigation.
In this study, we utilized the matrisome protein database as a reference [19] to identify 167 different proteins, aiming to determine the composition of the ECM derived from DPSCs. Glycoproteins were the most abundant, followed by regulator proteins, affiliated proteins, secreted proteins, collagens, and proteoglycans. Similarly, our previous study identified 225 matrisome proteins with comparable patterns in the matrisome category(8). In the periodontal ligament ECM, 119 matrisome proteins were found, with regulator proteins, glycoproteins, collagens, associated proteins, proteoglycans, and secretory proteins being the most prevalent (20). Conversely, the oral epithelium, as well as the early and late stages of oral tumors, exhibited 76, 63, and 43 matrisome proteins, respectively, with collagens, glycoproteins, proteoglycans, regulator proteins, affiliated proteins, and secreted proteins showing the greatest enrichment (21). These findings collectively suggest that the distinct ECM composition of each cell type or tissue directly influences its function.
The enrichment protein analysis of the ECM derived from DPSCs represents a crucial step in understanding the composition and functional significance of this specialized microenvironment. This analysis has provided valuable insights into the repertoire of ECM proteins, including structural proteins such as collagen, glycoproteins, and proteoglycans (22). The significant enrichment of proteins related to cellular components in the gene ontology analysis indicated that ECM-derived proteins are involved in building and maintaining various structural elements within cells (23). The number of proteins identified through gene ontology enrichment was noted in cellular components, cellular functions, and biological processes. Similarly, protein‒protein interactions were observed in collagen synthesis, collagen formation, ECM organization, laminin interactions, and the basement membrane. The recognition of laminin by integrin receptors is one of the most essential receptor types that mediates cell attachment to ECM components (24). Our KEGG pathway analysis revealed ECM receptor interactions, which may encompass signal transduction, cell adhesion, migration, and intracellular signaling pathways (2). Taken together, these results suggest that dECM functions are essential for cellular responses to the ECM environment and influence cell behavior.
The ECM controls cell behaviors during development, growth, and tissue regeneration (25). ECM component compositions regulate tissue stiffness, elasticity, and permeability (26). The current study demonstrated that the utilization of dECM substantially enhanced the adhesion, migration, and proliferation of gingival fibroblasts. Similarly, dECM derived from various sources of MSCs, such as adipose tissue (27), bone marrow (BM) (28-30), cartilage (31) and the umbilical cord (32), has been found to improve cell adhesion, migration, and proliferation. In addition, dECM derived from human periodontal ligament stem cells (hPDLSCs) promoted DPSC adhesion and spreading, as indicated by the strong expression of a focal adhesion protein (vinculin) and cell proliferation(33). However, the lack of significant effects on colony-forming ability raises interesting questions and considerations. A previous study reported that the colonies of adipose stem cells were larger and had greater cell density when cultured on ECM derived from BM. Compared with cells at late passages (passage 5), cells at lower passages (passage 3) exhibited greater colony-forming ability (29). Compared with cells cultured on medium without serum, cells cultured on BM-ECM in the presence of serum at passage 1 had significantly greater colony-forming units and proliferation rates (34). These findings suggest that a unique microenvironment promotes the colony-forming capacity and proliferation of MSCs while preserving their stem cell characteristics. Therefore, DPSC-derived dECM may enhance individual cell adhesion, migration, and proliferation; however, it may not substantially impact the ability of gingival fibroblasts to form colonies from single cells. The enhancement of gingival fibroblast attachment by dECM derived from DPSCs is a significant finding with implications for oral tissue regeneration. Gingival fibroblasts play a crucial role in maintaining gingival integrity and wound healing in the oral cavity (35).
The presence of specific ECM proteins, such as fibronectin and collagen, provides adhesive cues for cells, facilitating their attachment to the ECM and surrounding tissues. The identification of signaling focal contacts mediated by integrin beta3 (ITGβ3) has revealed its role in facilitating cell adhesion and migration, as well as its ability to mediate ECM-derived physical cues from bone marrow to modify the activity of hematopoietic stem and progenitor cells (HSPCs) (36). Together, laminin-binding integrins encourage epithelial cell attachment and proliferation(37). Moreover, the specific binding of ITGβ3 to the fibronectin ligand promoted the long-term repopulation of stem cells (38). Correspondingly, greater PDL cell proliferation and migration were triggered by the presence of fibronectin (39). Consistently, our results showed that ITGB3 and FN1 mRNA expression was significantly upregulated in gingival fibroblasts cultured on dECM derived from DPSCs.
WNT signaling pathways promote pulp healing, dentin repair, and stemness-related epigenetic regulation (40). Wnt/β-catenin signaling is activated during dental pulp healing and repair (41). Activation of this pathway can promote tissue regeneration, making it a crucial target for studies related to wound healing and regeneration (42). Our results demonstrated that CTNNB1 (the gene name for β-catenin) mRNA expression increased in gingival fibroblasts cultured on dECM derived from DPSCs. Similarly, it has also been reported that MSCs seeded on dECM derived from urine-derived stem cells enhanced chondrogenic differentiation via the WNT signaling pathway (43). Taken together, these findings suggest that interactions between the laminin basement dECM and β3 integrin could play a role in the adhesion, migration, and proliferation of gingival fibroblasts on the dECM, possibly involving the WNT signaling pathway. This finding highlights the complex interplay between the ECM microenvironment and cellular signaling and its relevance for tissue regeneration and wound healing in the oral cavity. Further research is necessary to elucidate the specific molecular pathways and mechanisms involved in gingival fibroblast adhesion, migration, and proliferation induced by DPSC-derived dECM and to explore their clinical applications in the context of oral health and regenerative medicine.