Exosome from adipose-derived stromal vascular fraction enhances the proliferation of human dental pulp stem cells

Abstract

Objectives:

In regenerative dentistry, cell proliferation is crucial for tissue repair and immune response modulation, essential for successful regeneration. The adipose-derived stromal vascular fraction (AD-SVF) shows promise in tissue engineering as an autologous therapy. AD-SVF exosomes, cell-free and resilient in ischemic conditions, offer an ethical and hopeful strategy for dental tissue regeneration and wider regenerative medicine use. Although the enhancement of human dental pulp stem cells (hDPSCs) migratory abilities by AD-SVF exosomes is known, their impact on hDPSC proliferation requires further examination.

Aims:

This study explores how AD-SVF exosomes influence hDPSC proliferation.

Methods:

AD-SVF exosomes, isolated using size exclusion chromatography and characterized through flow cytometry and nanoparticle tracking analysis, were used to treat hDPSCs at varying concentrations. Proliferation was assessed with the cell counting kit-8 assay. Statistical analysis involved one-way ANOVA, post hoc LSD testing, with significance at 0.05.

Results and Conclusion:

Isolated AD-SVF exosomes, averaging 103 ± 24 nm, expressed CD9+ and CD63+ markers. The study revealed increased hDPSC proliferation on the 5^th day post low-exosome treatment, indicating a positive association between AD-SVF exosomes and cell proliferation. These results highlight the potential of AD-SVF exosomes to enhance hDPSC proliferation for dental pulp and broader tissue regeneration.

INTRODUCTION

Regenerative dentistry has seen significant advancements, with a focus on tissue regeneration and the potential of exosomes from various sources to enhance dental stem cell function.[1] Previous research has shown that exosomes from adipose tissue, particularly Adipose-Derived Stromal Vascular Fraction (AD-SVF) exosomes, can boost the migratory capacity of human dental pulp stem cells (hDPSCs).[2] Dental pulp regeneration is challenging due to its unique location, leading to issues like ischemia and inflammation. AD-SVF, with its high survival rate in ischemic environments, offers a promising solution for tissue engineering.[3] This study aims to investigate the impact of AD-SVF exosomes on hDPSCs proliferation, addressing a gap in current research and potentially advancing therapeutic approaches in regenerative dentistry.

METHODS

Approval for this in vitro research was granted by the ethical review board at the Faculty of Dentistry, University of Indonesia (Ethical Approval No. 32/FKGUI/VIII/2023; Protocol No. 070530623). All adult subjects provided informed consent in accordance with the pertinent protocols. The AD-SVF Exosome employed in this study was acquired from the Dermama Laboratory in Surakarta, Indonesia, and isolated through size exclusion chromatography utilizing an automatic fraction collector Izon® qEV2 70 nm. Characterization was done via nanoparticle tracking analysis (NTA) with the ViewSizer 3000, enabling the measurement of size and concentration.[2]

Preparation of human dental pulp stem cells

In this investigation, hDPSCs were extracted from six individuals aged between 18 and 25 years, devoid of systemic illnesses or habits of smoking/alcohol consumption. The cells were sourced from intact third molars and premolars extracted for orthodontic reasons. Then cultured at Prodia Stem Cells Laboratory (Jakarta, Indonesia) at 37°C with 5% CO2 until 80% confluence (passages 3–4). After 24 h of nutrient deprivation in DMEM with 1% fetal bovine serum (FBS), they were treated with AD-SVF Exosomes. Flow cytometry confirmed the presence of mesenchymal stem cells (MSCs) markers CD90+, CD105+, CD73+, and the absence of lineage markers (LinNeg).

Proliferation assay

The proliferation of hDPSCs was assessed using the cell counting kit-8 (CCK-8) (Dojindo, Japan) as per the manufacturer’s instructions. hDPSCs (5 × 10³ cells/well) were seeded in 96-well plates and incubated overnight at 37°C. They were then cultured in medium with AD-SVF Exosome at concentrations of 0.1%, 1%, 5%, and 10%, while the control had medium only. At days 1, 3, and 5, 10 μL of CCK-8 solution was added to each well and incubated for 2 h at 37°C. Absorbance was measured at 450 nm using a microplate reader (Biobase, China).

Statistical analysis

Statistical analysis was performed using SPSS 29.0 and data visualization with GraphPad Prism 10 (GraphPad Software, Boston, Massachusetts, USA). Data are presented as mean ± standard deviation, with each experiment replicated at least three times. One-way ANOVA and LSD post hoc tests were used for comparisons among groups, with P < 0.05 considered significant.

RESULTS

Characterization of human dental pulp stem cells and adipose-derived stromal vascular fraction exosome

In this study, the qualitative assessment and expression of MSC markers in hDPSCs [Figure 1a-c] reaffirm the MSC nature of the cultured hDPSCs. Furthermore, the findings indicate a homogeneous nature of hDPSCs, characterized by forming loosely arranged colonies with distinctive spindle-shaped morphology. Through NTA imaging and analysis [Figure 1d and e], the AD-SVF Exo displayed a size distribution ranging from approximately 30–150 nm, with a specific diameter of 103 ± 24 nm and a particle concentration of 1.6 × 10⁸ particles/ml. Furthermore, specific marker proteins CD9+ and CD63+ were identified in the AD-SVF Exosome at a relative abundance of 90.9% through specific immunophenotyping using flow cytometry.

Figure 1.

Characterization of hDPSCs and AD-SVF Exosomes. hDPSCs at passages P3–P4 (a) and post-24-hour starvation (b). Flow cytometry confirmed mesenchymal markers: CD90+ (98%), CD105+ (99.7%), CD73+ (99.4%), and LinNeg (0.5%) (c). NTA images of AD-SVF Exosomes (d) with particle size and concentration analysis. Immunophenotyping showed CD63+/CD9+ markers (e). AD-SVF: Adipose-derived stromal vascular fraction, FITC: Fluorescein isothiocyanate

Proliferation of human dental pulp stem cells

The comparison of proliferation among groups indicates that all treatment AD-SVF Exosome concentrations (0.1%, 1%, 5%, and 10%) led to a notable significant increase in proliferation compared to the control group over (P < 0.001) distinct observation periods [Figure 2a and Table 1] with the most pronounced hDPSC proliferation observed on day 5. Specifically, LSD post hoc analysis identified significant variations among all concentrations. The 1% AD-SVF concentration was most effective on days 1 and 5, while the 0.1% concentration showed the highest impact on day 3. By day 5, all concentrations significantly enhanced proliferation compared to the control, though there were no significant differences between the various AD-SVF exosome concentrations. The highest proliferation occurred with 1% application of AD-SVF Exosomes on days 1 and 5, and with 0.1% on day 3. The proliferation of hDPSCs in response to each concentration of AD-SVF Exosome on every distinct observation period [Figure 2b and Table 1] also shows statistically significant differences across all exosome concentrations (P < 0.001).

Figure 2.

Impact of adipose-derived stromal vascular fraction (AD-SVF) exosomes on hDPSCs Proliferation. AD-SVF exosome treatment significantly enhanced hDPSCs proliferation at all concentrations (0.1%, 1%, 5%, and 10%) compared to the control (a). Proliferation increased progressively from day 1, peaking on day 5 across all concentrations (b). hDPSCs: Human dental pulp stem cells, AD-SVF: adipose-derived stromal vascular fraction

Table 1.

Proliferation assessment of human dental pulp stem cells following treatment with 0.1%, 1%, 5%, and 10% adipose-derived stromal vascular fraction exosomes across 1, 3, and 5 days of observation

Culture media	OD value at 450 nm			P
	Day 1, mean±SD	Day 3, mean±SD	Day 5, mean±SD
Control	1.939±0.082	3.33±0.132	4.44±0.326
0.1% AD-SVF Exosome	3.202±0.161	6.714±0.235	7.55±0.47	<0.001*
1% AD-SVF Exosome	3.515±0.182	6.362±0.667	7.723±0.345	<0.001*
5% AD-SVF Exosome	3.482±0.175	6.33±0.142	7.69±0.41	<0.001*
10% AD-SVF Exosome	3.261±0.209	5.954±0.164	7.413±0.254	<0.001*
P	<0.001*	<0.001*	<0.001*

*One-way ANOVA test, P<0.05. AD-SVF: Adipose-derived stromal vascular fraction, SD: Standard deviation, OD: Optical density

DISCUSSION

Cell proliferation, differentiation, and maturation are essential to cell growth and development.[4] Cells divide and reproduce during cell proliferation, with one daughter cell maintaining its stem cell characteristics while the other differentiates. Growth factors are crucial in initiating the cell cycle and driving cell proliferation. The overall cell cycle takes approximately 24 h.[5] Cell proliferation serves as a fundamental defense mechanism, aiding in the limitation of inflammation and accelerating tissue repair and regeneration.[6] Therefore, augmenting cell proliferation and enhancing cell survival contributes to safeguarding the pulp against inflammation.

Exosomes have paracrine effects on tissue regeneration by transmitting information to damaged cells and displaying activities akin to their originating cells. Each cell source has unique cargo that influences therapeutic benefits and outcomes.[7] The International Society of Extracellular Vesicles requires the use of at least two distinct technologies for exosome identification.[8] This study utilizes flow cytometry to characterize surface proteins on AD-SVF exosomes and NTA for assessing particle size and concentration. This dual approach enhances the reliability and accuracy of the findings. Hence, this study employed flow cytometry (CD63+ and CD9+) for specific surface protein characterization of AD-SVF Exosomes, confirming their exosomal nature. The content of exosomes is dynamic and highly dependent on the cell origin and physiological status. NTA results clearly demonstrate that AD-SVF exosomes possess a mean diameter of 103 ± 24 nm and a high particle concentration of 1,6 × 10⁸ particles/ml [Figure 1e]. These measurements not only comply with the established exosome size criteria of 30–150 nm but also underscore their role as novel carriers for intercellular communication to influencing cellular biological behaviors.[9]

Our present study evaluates the impact of AD-SVF exosomes on the regeneration of hDPSCs, mainly focusing on their influence on proliferation. In our proliferation assays [Figure 2], we observed a significant increase in hDPSC proliferation at specific concentrations of AD-SVF exosomes. Notably, lower concentrations (especially 0.1% and 1%) were linked to remarkable increases in cellular proliferation time-dependently. It was aligning with previous findings regarding the positive effects of insulin-like growth factor and fibroblast growth factor (FGF) on hDPSC proliferation.[10] A parallel observation was noted with concentrated growth factors, highlighting a rapid proliferation rate in human-induced pluripotent stem cells.[11] Similarly, the regenerative potential observed in injectable platelet-rich fibrin and leukocyte-platelet-rich fibrin), reflecting enhanced proliferation and DPSC activation for tissue regeneration, resonates with our study’s focus on the proliferative benefits of AD-SVF exosomes.[12] This correlation reinforces the significance of AD-SVF exosomes in promoting hDPSC proliferation and underscores their potential in regenerative applications within the dental pulp environment.

The AD-SVF is composed of a diverse range of cellular components, growth factors, and microRNA (miRNAs) that collectively play crucial roles in paracrine signaling, differentiation, immunomodulation, angiogenesis, vascularization, extracellular matrix modulation, and inter-element interactions. The cellular components within AD-SVF include various progenitor cells, including ADSCs (Adipose-Derived Stem Cells), endothelial cells, and macrophages. These components contribute to the regenerative potential of AD-SVF through the secretion of growth factors such as VEGF (Vascular Endothelial Growth Factor), FGF, and HGF (Hepatocyte Growth Factor).[3,13] ADSCs, the primary element of AD-SVF (up to 80% of the total SVF population), underscore its role as an immunomodulator, anti-apoptotic, antifibrotic, and angiogenic agent. ADSCs enhance tissue regeneration and modulate the immune response through paracrine signaling, growth factor secretion, and interactions with other cells within AD-SVF.[3,14] The combined action of these cellular components, growth factors, and miRNAs within the AD-SVF underscores its significance in promoting tissue regeneration and overall physiological balance.

AD-SVF contains miRNAs that influence cellular functions and regeneration. These miRNAs come from various components in AD-SVF, such as ADSCs (e.g., miR-378, miR-221, and miR-320d), endothelial cells (miR-126 and miR-424), and macrophages (miR-146a and miR-125a).[3] AD-SVF miRNAs are associated with pulp inflammation, such as miR-146a, which is critical in enhancing cell proliferation,[15] decreased miR-221 indirectly regulating endothelial nitric oxide synthase expression, potentially impacting local vasodilation and cell proliferation within the dental pulp.[16] Despite these associations, this study has yet to confirm the actual activities of AD-SVF miRNAs related to dental pulp regeneration.

Previous research has shown that ADSC-Exosomes stimulate fibroblasts’ proliferation and migration through the activation of the wingless-related integration site/beta-catenin (WNT/β-catenin) and phosphoinositide-3-kinase-Akt (PI3K/AKT) signaling pathways.[17] The regulatory mechanisms in dental pulp proliferation involving FGF/FGF receptor-mitogen-activated protein kinase/extracellular signal-regulated kinase (FGF/FGFR-MEK/ERK) and WNT/β-catenin pathways are crucial for cell cycle control and cell renewal to enhance stem cell proliferation.[18] This evidence suggests a pivotal role of ADSC-Exosomes in tissue regeneration by promoting cellular proliferation. Additionally, enhancing FGF-2 mRNA expression is crucial for wound healing and cell proliferation.[19]

Dental pulp cell proliferation is also associated with FGF-2, which is significantly upregulated at the mRNA level within 24 h of exosome exposure.[20] Recent research also shows that DPSCs’ proliferation time on days 1, 3, 5, and 7 increases time-dependently by 50 μg/ml DPSC-Exos.[21] The initiation of the proliferation phase is attributable to the expression of FGF, which activates the MEK/ERK and canonical WNT pathways, potentially leading to transdifferentiation into odontoblasts, angiogenesis, or neurogenesis, depending on the distinctive requirements of the pulp.[18] FGF contributes significantly to DPSCs’ proliferation by inhibiting apoptosis and activating the ERK pathway, leading to enhanced cellular proliferation and migration through platelet-derived growth factor receptor beta/phosphorylated extracellular signal-regulated kinase (PDGFRb/p-Erk) and self-renewal through Akt and Erk1/2 pathways.[22] The significant increase in hDPSC proliferation observed in our study underscores the potential of AD-SVF Exosomes as a valuable asset in dental pulp regeneration.

The AD-SVF exosomes (AD-SVF exosomes) significantly enhance the proliferation of hDPSCs. While higher concentrations do not always equal increased efficacy, a concentration of 0.1% and 1% AD-SVF exosomes produced the most significant in vitro results concerning the enhancement of proliferation of hDPSCs. This research offers insights into tackling the regenerative challenges in hDPSCs within the domain of regenerative endodontics, emphasizing the essential role of a conducive microenvironment for dental pulp regeneration. The utilization of AD-SVF Exosome aligns well with the concept of providing a controlled environment conducive to both sterilization and regenerative processes, crucial for successful revitalization and regeneration in cases like immature teeth with fragile root walls.[23] This approach underscores the potential of exosome-based strategies in overcoming hurdles associated with regenerative endodontics, enhancing the prospects for effective treatment outcomes in dental pulp regeneration.[24] However, it is crucial to acknowledge certain limitations. This study, being conducted in vitro, may not fully represent the clinical scenario, and its specificity is confined to the particle size and concentration of AD-SVF Exosomes within this research. A more comprehensive analysis, encompassing a variety of markers and differentiation stages of regeneration, is necessary to validate these findings. Additionally, exploring in vivo studies to enhance our understanding. Future research should aim for a more comprehensive understanding of these aspects.

CONCLUSION

AD-SVF exosomes are pivotal in dental pulp regeneration and tissue engineering, by promoting hDPSCs proliferation. Their cell-free nature and resilience in ischemic conditions make them an ethical and promising solution for dental tissue regeneration and broader regenerative medicine applications.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was financially supported by the Indonesia Endowment Fund for Education Agency (Lembaga Pengelola Dana Pendidikan, LPDP) grant number 0001276/TRP/D/PDD-2020.