Jun 30, 2026
Effect of SHED-derived Secretome and Mineral Trioxide Aggregate on the regenerative potential and viability of inflamed Dental Pulp Cells : An Invitro Study
- ElShaymaa Ragab Shehab1,
- Prof. Dr. Ghada El-Hilaly Eid2,
- Dr. Marwa Magdy Saad Abbass3,
- Dr. Ghada ElWazan4
- 1Master's Degree Candidate, Department of Endodontics, Faculty of Dentistry, Cairo University;
- 2Professor of Endodontics, Department of Endodontics, Faculty of Dentistry, Cairo University;
- 3Professor of Oral Biology at the Faculty of Dentistry, German university in Cairo and Cairo University;
- 4Lecturer in Endodontics, Department of Endodontics, Faculty of Endodontics, Cairo University
- Cairo University
Protocol Citation: ElShaymaa Ragab Shehab, Prof. Dr. Ghada El-Hilaly Eid, Dr. Marwa Magdy Saad Abbass, Dr. Ghada ElWazan 2026. Effect of SHED-derived Secretome and Mineral Trioxide Aggregate on the regenerative potential and viability of inflamed Dental Pulp Cells : An Invitro Study. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmdkwbv3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 28, 2026
Last Modified: June 30, 2026
Protocol Integer ID: 319960
Keywords: regenerative endodontic therapy under inflammatory condition, inflamed dental pulp cell, regenerative potential of dental pulp, human dental pulp cell, dental pulp cell, enhancing regenerative endodontic therapy, gold standard in vital pulp therapy, mineral trioxide aggregate on the regenerative potential, vital pulp therapy, dental pulp, odontogenic potential, invitro study inflammation, mineral trioxide aggregate, lipopolysaccharide, derived secretome, angiogenesi, inflammation, odontogenic differentiation, secretome, inflammatory condition
Abstract
Inflammation adversely affects the regenerative potential of dental pulp by impairing cell viability, angiogenesis, and odontogenic differentiation. Although Mineral Trioxide Aggregate (MTA) is a gold standard in vital pulp therapy, its ability to modulate inflammation remains limited. This study aims to evaluate the effect of SHED-derived secretome, alone and in combination with MTA, on inflamed dental pulp cells. In this in vitro study, human dental pulp cells will be stimulated with lipopolysaccharide (LPS) to induce inflammation and divided into four groups: SHED-secretome, MTA, combined SHED-secretome + MTA, and LPS-only control. Biocompatibility will be assessed using the MTT assay, while angiogenic and odontogenic potentials will be evaluated through VEGF and DSPP gene expression. It is hypothesized that the combination therapy will yield superior regenerative outcomes. This approach may provide a more effective strategy for enhancing regenerative endodontic therapy under inflammatory conditions.
Guidelines
| The research will be submitted to the research ethics committee for review. After receiving the results and finishing the experiment, all the samples will be sterilized and discarded in a special incinerator under supervision of Microbiology Department-Cairo University. The informed consent given to patients whom we will extract the permeant third molars. (Refer to Appendix A) Parents of pediatric patients will be given an ascent and a consent form will be signed. |
Materials
| First: Sampling and Cell Isolation of SHED Using Explant Method 1- Sampling Collection Freshly extracted healthy human deciduous teeth (5–8 years old) will be collected from patients undergoing natural exfoliation or orthodontic extraction at the Department of Pediatric Dentistry, Cairo University, with informed parental consent and ethical approval. Samples will be transported in sterile PBS with 1% antibiotic–antimycotic solution at 4 °C and processed within 24 h (Miura et al., 2003)24. 2. Explant Culture and Cell Expansion Pulp tissues will be aseptically extracted, minced into small fragments (1–2 mm³), and placed in 25 cm² culture flasks containing complete culture medium (DMEM–low glucose, 20% FBS, 2 mM L‑glutamine, 100 U/mL penicillin–streptomycin). Cultures will be maintained at 37 °C, 5% CO₂ with medium changes every 2–3 days. Cells will be passaged at 80–90% confluence and used between passages 3–5 (Hendijani, 2017, Nardi & da Silva, 2008)25, 26. 3. Characterization SHED characterization will be confirmed by flow cytometry (positive for CD73, CD90, CD105; negative for CD34, CD45) (Yumkham et al., 2024)27. 4. Extraction of Secretome (Conditioned Medium) SHED are to be cultured in Dulbecco's modified Eagle medium (DMEM) until they reach the desired passage (usually 3-5th passage). Culture medium is replaced with a serum-free medium to prevent contamination of secretome with serum proteins. The conditioned medium (containing the secretome) is collected after 48 hours of incubation. It is then collected and clarified through sequential centrifugation first at 300 × g for 5 minutes to pellet cells, followed by 2,000 × g for 10–15 minutes to remove debris and apoptotic bodies. The supernatant is then filtered through a 0.22 μm low-protein-binding filter for sterilization and stored at -80 °C (Bastidas et al., 2023)28. Second: Isolation and culture of Human Dental Pulp Cells (DPCs) The explant method is to be used in the isolation of primary human dental pulp cells: Primary human DPCs will be isolated using the explant (outgrowth) method. After surface sterilization, pulp tissue will be removed, minced (~1 mm³), and cultured in growth medium under standard conditions (37 °C, 5% CO₂). After being confluent, cells will be subcultured using 0.25% trypsin‑EDTA and employed between passages 2–4 (Kearney et al., 2024)29. Third: Induction of LPS-Induced Inflammation Lipopolysaccharide (E. coli O111:B4) will be prepared at 1 mg/mL in sterile PBS, stored at −20 °C, and diluted to 0.1 μg/mL for use. DPCs will be seeded in 6‑well plates and treated with LPS for 48 h to establish a mild inflammation model (Wang et al., 2022)30. Fourth: Formation of MTA Elution/Extract ProRoot MTA (Dentsply) will be mixed at a 3:1 (w/w) Powder‑to‑liquid ratio. Set discs (6 mm × 2 mm) will be incubated for 7 days in serum‑free DMEM at 37 °C (3 cm²/mL ratio) following (ISO 10993‑12:2012)31 standards to obtain the extract. After filtration (0.22 μm), the MTA elute will be used undiluted (1:1) for subsequent assays (Ha et al., 2024, Ayoub et al., 2025)10,32. |
Before start
- Eligibility criteria**:
- Inclusion Criteria**
- SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth)**
- Tooth type: Deciduous teeth (primary dentition)
- Patient age: 5–8 years
- Extraction indication: Physiological shedding (natural exfoliation)
- Caries status: Caries-free
- Processing time: Within 1 hour of extraction
- Human DPCs Source (Dental Pulp Cells)**
- Tooth type: Non-carious third molars
- Patient age: 18–25 years
- Extraction indication: Routine extraction (orthodontic or prophylactic)
- Caries status: Non-carious
- Pulp condition: Healthy pulp tissue
- Processing time: Within 1 hour of extraction
- Exclusion Criteria**
- SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth)**
- Active caries, restorations, or caries history
- Pulpitis or pulp necrosis
- Periapical pathology
- Premature or trauma-induced extraction
- Orthodontically mobilized teeth
- Systemic disease affecting dental tissues
- Active oral or systemic infections
- Long-term corticosteroid use, chemotherapy, or bisphosphonate therapy
- Human DPCs Source (Dental Pulp Cells)**
- Caries, pulpitis, pulp necrosis, or previous pulp therapy
- Periapical pathology or periodontal disease affecting dental tissues
- Extraction due to pathology
- Systemic disease affecting dental tissues
- Active oral or systemic infections
- Long-term corticosteroid use, chemotherapy, or bisphosphonate therapy
III. Methods
MTA alone and those under LPS-induced in...
Trial Design: In vitro
Allocation Ratio: 1:1:1
Framework: superiority
III-A) Samples, Intervention and Outcomes
III-7. A) Calculated sample size: Regarding the primary outcome (cell viability), sample size estimation was performed using previously published data from (Ha et al., 2024) which evaluated the effects of hydraulic calcium silicate cements.
A large effect size (d = 1.29) was estimated from the reported cell viability outcomes of the reference study. Using G*Power software version 3.1 (Heinrich Heine University, Düsseldorf, Germany), with a significance level (α) of 0.05, study power of 80%, and two-sided hypothesis testing, the minimum required sample size was calculated to be 3 samples per group. However, because the present study involves LPS-induced inflamed dental pulp cells, which may demonstrate greater biological variability than non-inflamed cell models, the sample size was increased to 5 replicates per group (total sample size of 20) to improve the reliability, reproducibility, and statistical validity of the results.
III-8.A) Description of study sample: In this in-vitro study, the study sample consists of SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth) and human dental pulp derived cells (DPCs) from extracted third molars.
Stem Cells from Human Exfoliated Deciduous Teeth (SHED) are going to be used to extract the secretome which will be used in the intervention groups. They will be extracted from non-carious, physiologically shedding deciduous teeth, collected from healthy donors following assent and informed consent and ethical approval. Extracted SHED will not be used in any clinical studies, and if stored it will only be used in the present in-vitro study.
Human dental pulp derived cells (DPCs) will be the tested sample onto which the MTA, Secretome and their combination will be applied. They are cultured under standardized laboratory conditions. Human DPCs are to be isolated from extracted human third molars collected from healthy donors following informed consent and ethical approval. Extracted human DPCs will not be used in any clinical studies, and if stored will only be used in the present in-vitro studies.
Eligibility criteria will be applied to the cell source and experimental conditions, as follows; Table 2:
Table 2: The Eligibility criteria of SHED and human DPCs.
Inclusion Criteria
SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth)
• Tooth type: Deciduous teeth (primary dentition)
• Patient age: 5–8 years
• Extraction indication: Physiological shedding (natural exfoliation)
• Caries status: Caries-free
• Processing time: Within 1 hour of extraction
Human DPCs Source (Dental Pulp Cells)
• Tooth type: Non-carious third molars
• Patient age: 18–25 years
• Extraction indication: Routine extraction (orthodontic or prophylactic)
• Caries status: Non-carious
• Pulp condition: Healthy pulp tissue
• Processing time: Within 1 hour of extraction
• Cell cultures will be confirmed free of contamination, exhibited 3e90% viability, and will be used between passages 3–5
• Cell cultures will be confirmed free of contamination, exhibited 3e90% viability and will be used between passages 2–4
Exclusion Criteria
SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth)
• Active caries, restorations, or caries history
• Pulpitis or pulp necrosis
• Periapical pathology
• Premature or trauma-induced extraction
• Orthodontically mobilized teeth
• Systemic disease affecting dental tissues
• Active oral or systemic infections
• Long-term corticosteroid use, chemotherapy, or bisphosphonate therapy
Human DPCs Source (Dental Pulp Cells)
• Caries, pulpitis, pulp necrosis, or previous pulp therapy
• Periapical pathology or periodontal disease affecting dental tissues
• Extraction due to pathology
• Systemic disease affecting dental tissues
• Active oral or systemic infections
• Long-term corticosteroid use, chemotherapy, or bisphosphonate therapy
III-8. B) Sample Preparation
Preparation will include four main steps: First: Sampling and Cell Isolation of SHED Second: Isolation and culture of Human Dental Pulp Cells (DPCs), Third: Induction of LPS Induced Inflammation of human DPCs, Fourth: Formation of MTA Elution/Extract
First: Sampling and Cell Isolation of SHED:
1. Sampling
2. Cell Outgrowth and expansion
3. Characterization
4. Secretome Extraction
Second: Isolation and culture of human Dental Pulp Cells (DPCs)
Third: Induction of LPS Induced Inflammation of DPCs
Fourth: Formation of MTA Elution/Extract
| First: Sampling and Cell Isolation of SHED Using Explant Method 1- Sampling Collection Freshly extracted healthy human deciduous teeth (5–8 years old) will be collected from patients undergoing natural exfoliation or orthodontic extraction at the Department of Pediatric Dentistry, Cairo University, with informed parental consent and ethical approval. Samples will be transported in sterile PBS with 1% antibiotic–antimycotic solution at 4 °C and processed within 24 h (Miura et al., 2003)24. 2. Explant Culture and Cell Expansion Pulp tissues will be aseptically extracted, minced into small fragments (1–2 mm³), and placed in 25 cm² culture flasks containing complete culture medium (DMEM–low glucose, 20% FBS, 2 mM L‑glutamine, 100 U/mL penicillin–streptomycin). Cultures will be maintained at 37 °C, 5% CO₂ with medium changes every 2–3 days. Cells will be passaged at 80–90% confluence and used between passages 3–5 (Hendijani, 2017, Nardi & da Silva, 2008)25, 26. 3. Characterization SHED characterization will be confirmed by flow cytometry (positive for CD73, CD90, CD105; negative for CD34, CD45) (Yumkham et al., 2024)27. 4. Extraction of Secretome (Conditioned Medium) SHED are to be cultured in Dulbecco's modified Eagle medium (DMEM) until they reach the desired passage (usually 3-5th passage). Culture medium is replaced with a serum-free medium to prevent contamination of secretome with serum proteins. The conditioned medium (containing the secretome) is collected after 48 hours of incubation. It is then collected and clarified through sequential centrifugation first at 300 × g for 5 minutes to pellet cells, followed by 2,000 × g for 10–15 minutes to remove debris and apoptotic bodies. The supernatant is then filtered through a 0.22 μm low-protein-binding filter for sterilization and stored at -80 °C (Bastidas et al., 2023)28. Second: Isolation and culture of Human Dental Pulp Cells (DPCs) The explant method is to be used in the isolation of primary human dental pulp cells: Primary human DPCs will be isolated using the explant (outgrowth) method. After surface sterilization, pulp tissue will be removed, minced (~1 mm³), and cultured in growth medium under standard conditions (37 °C, 5% CO₂). After being confluent, cells will be subcultured using 0.25% trypsin‑EDTA and employed between passages 2–4 (Kearney et al., 2024)29. Third: Induction of LPS-Induced Inflammation Lipopolysaccharide (E. coli O111:B4) will be prepared at 1 mg/mL in sterile PBS, stored at −20 °C, and diluted to 0.1 μg/mL for use. DPCs will be seeded in 6‑well plates and treated with LPS for 48 h to establish a mild inflammation model (Wang et al., 2022)30. Fourth: Formation of MTA Elution/Extract ProRoot MTA (Dentsply) will be mixed at a 3:1 (w/w) Powder‑to‑liquid ratio. Set discs (6 mm × 2 mm) will be incubated for 7 days in serum‑free DMEM at 37 °C (3 cm²/mL ratio) following (ISO 10993‑12:2012)31 standards to obtain the extract. After filtration (0.22 μm), the MTA elute will be used undiluted (1:1) for subsequent assays (Ha et al., 2024, Ayoub et al., 2025)10,32. |
Protocol references
1. El-Sayed et al., 2019
2. Qiao et al., 2025
3. Asgary 26 Eghbal, 2020
4. Parirokh et al., 2011
5. Weekate et al., 2021
6. Chung et al., 2019
7. Suresh et al., 2025
8. Pay e3o et al., 2025
9. Costa et al., 2022
10. Ha et al., 2024
11. Grieco et al., 2025
12. Kim et al., 2025
13. Kim et al., 2018
14. Wang et al., 2021
15. Wang et al., 2023
16. Sarra et al., 2021
17. Silva et al., 2023
18. Mohd Nor et al., 2023
19. Akhlaghi 26 Khademi, 2015
20. Cervino et al., 2020
21. Komora et al., 2024
Acknowledgements
- Aim of the study**: to determine whether the incorporation of secretome derived from stem cells from human exfoliated deciduous teeth (SHED) enhances the biocompatibility, angiogenic, and odontogenic potential of mineral trioxide aggregate when applied to LPS-induced inflamed dental pulp-derived cells as compared to MTA alone and secretome alone in an vitro model mimicking pulpitis.
- Objective**: Regarding MTA, secretome, and their combination assess: First: Biocompatibility on human DPCs. Second: angiogenic potential by analyzing VEGF gene expression, Third: odontogenic differentiation through DSPP gene expression analysis.
- PICO**
- P: Population**: Lipopolysaccharide (LPS)-Stimulated human dental pulp derived cells
- I: Intervention**:
- I1**: SHED-derived secretome alone; for treating LPS-induced inflamed human dental pulp derived cells
- I2**: SHED-derived secretome in combination with mineral trioxide aggregate; for treating LPS-induced inflamed human dental pulp derived cells
- C: Comparator**: Mineral trioxide aggregate alone (representing current standard bioactive material); for treating LPS-induced inflamed human dental pulp derived cells
- O. Outcomes, Table 1**
- O1**: Biocompatibility
- O2**: Angiogenic potential (VEGF gene expression)
- O3**: Odontogenic differentiation (DSPP gene expression)
- Null Hypothesis (H0)**: There is no significant difference in biocompatibility, angiogenic potential, odontogenic differentiation, between human DPCs treated with MTA alone and those treated with Secretome or MTA combined with Secretome under LPS-induced inflammatory conditions.
- Trial Design**: In vitro comparative study.
- Allocation Ratio**: 1:1:1
- Framework**: superiority
- Outcomes**:
- Primary outcome Biocompatibility**: The ability of the tested material to maintain or enhance cell viability of human DPCs without inducing cytotoxic effects. Assessment Method**: MTT reduction Assay (Kumar et al, 2018)^^12^^. Unit of Measurement**: Optical density at 570 nm.
- Secondary outcome Odontogenic Potential**: The ability of treated human DPCs to acquire an odontoblast-like phenotype characterized by the expression of dentin specific genes e.g., DSPP. Assessment Method**: DSPP gene expression (Ha et al, 2024)^^10^^. Unit of Measurement**: Relative gene expression (2^-ΔΔCt).
- Secondary outcome Angiogenic potential**: The capacity of treated human DPCs to promote angiogenesis by upregulating VEGF to support pulp vascularization and tissue repair. Assessment Method**: VEGF gene expression (Gomez-Sosa et al 2021)^^23^^. Unit of Measurement**: Relative gene expression (2^-ΔΔCt).
- Methods**:
- III-A) Samples, Intervention and Outcomes**
- III-7. A) Calculated sample size**: Regarding the primary outcome (cell viability), sample size estimation was performed using previously published data from (Ha et al., 2024)^^10^^, which evaluated the effects of hydraulic calcium silicate cements and stem cell-derived secretome on human dental pulp stem cells under comparable experimental conditions. Although the referenced study was conducted on non-inflamed cells, it was selected due to the similarity in cell type, intervention, and outcome assessment method (cell viability assay). The inflammatory experimental model used in the present study was further supported by (Wang et al. 2022)^^15^^, which investigated lipopolysaccharide (LPS)-induced inflamed dental pulp cells under conditions relevant to the current study design.
- A large effect size (d = 1.29) was estimated from the reported cell viability outcomes of the reference study. Using G*Power software version 3.1 (Heinrich Heine University, Düsseldorf, Germany), with a significance level (α) of 0.05, study power of 80%, and two-sided hypothesis testing, the minimum required sample size was calculated to be 3 samples per group. However, because the present study involves LPS-induced inflamed dental pulp cells, which may demonstrate greater biological variability than non-inflamed cell models, the sample size was increased to 5 replicates per group (total sample size of 20) to improve the reliability, reproducibility, and statistical validity of the results.
- III-8.A) Description of study sample**:
- In this in-vitro study, the study sample consists of SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth) and human dental pulp derived cells (DPCs) from extracted third molars.
- Stem Cells from Human Exfoliated Deciduous Teeth (SHED) are going to be used to extract the secretome which will be used in the intervention groups. They will be extracted from non-carious, physiologically shedding deciduous teeth, collected from healthy donors following assent and informed consent and ethical approval. Extracted SHED will not be used in any clinical studies, and if stored it will only be used in the present in-vitro study.
- Human dental pulp derived cells (DPCs) will be the tested sample onto which the MTA, Secretome and their combination will be applied. They are cultured under standardized laboratory conditions. Human DPCs are to be isolated from extracted human third molars collected from healthy donors following informed consent and ethical approval. Extracted human DPCs will not be used in any clinical studies, and if stored will only be used in the present in-vitro studies.
- Eligibility criteria will be applied to the cell source and experimental conditions, as follows; Table 2:
- Table 2: The Eligibility criteria of SHED and human DPCs.**
- Inclusion Criteria**
- SHED Source (Stem Cells from Human Exfoliated Deciduous Teeth)**
- Tooth type: Deciduous teeth (primary dentition)
- Patient age: 5–8 years
- Extraction indication: Physiological shedding (natural exfoliation)
- Caries status: Caries-free
- Processing time: Within 1 hour of extraction
- Human DPCs Source (Dental Pulp Cells)**
- Tooth type: Non-carious third molars
- Patient age: 18–25 years
- Extraction indication: Routine extraction (orthodontic or prophylactic)
- Caries status: Non-carious
- Pulp condition: Healthy pulp tissue
- Processing time: Within 1 hour of extraction
- III-8.B) Sample Preparation**:
- Preparation will include four main steps: First: Sampling and Cell Isolation of SHED Second: Isolation and culture of Human Dental Pulp Cells (DPCs), Third: Induction of LPS Induced Inflammation of human DPCs, Fourth: Formation of MTA Elution/Extract
- First: Sampling and Cell Isolation of SHED:
1. Sampling
2. Cell Outgrowth and expansion
3. Characterization
4. Secretome Extraction
- Second: Isolation and culture of human Dental Pulp Cells (DPCs)
- Third: Induction of LPS Induced Inflammation of DPCs
- Fourth: Formation of MTA Elution/Extract