{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T13:26:40Z","timestamp":1780579600414,"version":"3.54.1"},"reference-count":54,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T00:00:00Z","timestamp":1761868800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>This study aimed to assess the biocompatibility and bioactivity of three different silver nanoparticles (AgNPs) and calcium hydroxide [Ca(OH)2] mixtures against human dental pulp stem cells (hDPSCs). hDPSCs were treated with one of the following medicaments: 2 nm mixture, 5 nm mixture, 10 nm mixture, Ca(OH)2 alone, and triple antibiotic paste (TAP). Cell viability was evaluated using the Cell Counting Kit-8 and LIVE\/DEAD Viability\/Cytotoxicity Kit. Reactive oxygen species (ROS) were quantified using the 2\u2032,7\u2032-dichlorofluorescein diacetate redox probe. Transforming growth factor (TGF)-\u03b21, interleukin (IL)-1\u03b2, tumor necrosis factor (TNF)-\u03b1&gt;, and alkaline phosphatase (ALP) were quantified using enzyme-linked immunosorbent assays. Mineralization was assessed using Alizarin Red S staining. Data were compared across groups using the Kruskal\u2013Wallis test and within groups using the\u00a0Wilcoxon signed-rank test (p &lt; 0.05). Ca(OH)2 alone and the 10 nm mixture demonstrated the highest cell viability and lowest ROS release (p &lt; 0.05), while the 2 nm and 5 nm mixtures resulted in decreased viability and significant morphological distortion of the cells. Ca(OH)2 alone and the 10 nm mixture comparably demonstrated the highest production of anti-inflammatory cytokine TGF-\u03b21 (p &lt; 0.05), the lowest production of proinflammatory cytokines IL-1\u03b2 and TNF-\u03b1 (p &lt; 0.05), and the highest ALP release and mineralization (p &lt; 0.05). Within the limitations of this in vitro study, Ca(OH)2 alone and the 10 nm mixture improved hDPSCs\u2019 viability, proliferation, differentiation, and mineralization. Both illustrated a significantly higher anti-inflammatory response by the residing stem cell population.<\/jats:p>","DOI":"10.3390\/ijms262110604","type":"journal-article","created":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T07:29:42Z","timestamp":1761895782000},"page":"10604","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Size-Dependent Bioactivity of Silver Nanoparticles and Calcium Hydroxide Mixtures Against hDPSCs: An In Vitro Study"],"prefix":"10.3390","volume":"26","author":[{"given":"Ghazal","family":"Fakeeha","sequence":"first","affiliation":[{"name":"Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Riyadh 11595, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8663-6426","authenticated-orcid":false,"given":"Lama","family":"Al-Zamil","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh 12372, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3057-1851","authenticated-orcid":false,"given":"Manikandan","family":"Muthurangan","sequence":"additional","affiliation":[{"name":"Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh 12372, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sayed","family":"Auda","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh 12372, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hanan","family":"Balto","sequence":"additional","affiliation":[{"name":"Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Riyadh 11595, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1016\/j.cden.2009.12.006","article-title":"Treatment of the Immature Tooth with a Non\u2013Vital Pulp and Apical Periodontitis","volume":"54","author":"Trope","year":"2010","journal-title":"Dent. Clin. N. Am."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"S46","DOI":"10.1016\/j.joen.2017.06.010","article-title":"Microbial Factors and Antimicrobial Strategies in Dental Pulp Regeneration","volume":"43","author":"Fouad","year":"2017","journal-title":"J. Endod."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1273","DOI":"10.1016\/j.joen.2020.05.011","article-title":"Comparison of the Biocidal Efficacy of Sodium Dichloroisocyanurate and Calcium Hydroxide as Intracanal Medicaments over a 7-Day Contact Time: An Ex Vivo Study","volume":"46","author":"Chan","year":"2020","journal-title":"J. Endod."},{"key":"ref_4","unstructured":"American Association of Endodontists (2025, October 13). Clinical Considerations for a Regenerative Endodontic Procedure. Available online: https:\/\/www.aae.org\/specialty\/wp-content\/uploads\/sites\/2\/2021\/08\/ClinicalConsiderationsApprovedByREC062921.pdf."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1097\/00004770-200210000-00003","article-title":"Biofilm Formation in Medicated Root Canals","volume":"28","author":"Distel","year":"2002","journal-title":"J. Endod."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.1016\/j.joen.2016.05.021","article-title":"Antibacterial Nanoparticles in Endodontics: A Review","volume":"42","author":"Shrestha","year":"2016","journal-title":"J. Endod."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1689","DOI":"10.1016\/j.joen.2020.07.001","article-title":"Combined Effect of a Mixture of Silver Nanoparticles and Calcium Hydroxide against Enterococcus faecalis Biofilm","volume":"46","author":"Balto","year":"2020","journal-title":"J. Endod."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1573","DOI":"10.1016\/j.jdent.2015.08.012","article-title":"Antibiofilm efficacy of silver nanoparticles as a vehicle for calcium hydroxide medicament against Enterococcus faecalis","volume":"43","author":"Afkhami","year":"2015","journal-title":"J. Dent."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1294","DOI":"10.1016\/j.joen.2022.07.008","article-title":"Antibiofilm Efficacy of Silver Nanoparticles Alone or Mixed with Calcium Hydroxide as Intracanal Medicaments: An Ex-Vivo Analysis","volume":"48","author":"AlGazlan","year":"2022","journal-title":"J. Endod."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1213","DOI":"10.1016\/j.identj.2024.08.007","article-title":"The Impact of Silver Nanoparticles\u2019 Size on Biofilm Eradication","volume":"75","author":"Fakeeha","year":"2024","journal-title":"Int. Dent. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"S95","DOI":"10.1016\/j.joen.2017.07.012","article-title":"Microbial Modulation of Stem Cells and Future Directions in Regenerative Endodontics","volume":"43","author":"Diogenes","year":"2017","journal-title":"J. Endod."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.toxlet.2010.12.001","article-title":"Silver nanoparticles: Evaluation of DNA damage, toxicity and functional impairment in human mesenchymal stem cells","volume":"201","author":"Hackenberg","year":"2011","journal-title":"Toxicol. Lett."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2058","DOI":"10.3762\/bjnano.5.214","article-title":"Effect of silver nanoparticles on human mesenchymal stem cell differentiation","volume":"5","author":"Sengstock","year":"2014","journal-title":"Beilstein J. Nanotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Manke, A., Wang, L., and Rojanasakul, Y. (2013). Mechanisms of Nanoparticle-Induced Oxidative Stress and Toxicity. Biomed. Res. Int., 2013.","DOI":"10.1155\/2013\/942916"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"319","DOI":"10.3109\/17435390.2010.483745","article-title":"Impact of silver nanoparticles on human cells: Effect of particle size","volume":"4","author":"Liu","year":"2010","journal-title":"Nanotoxicology"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"13608","DOI":"10.1021\/jp712087m","article-title":"Unique Cellular Interaction of Silver Nanoparticles: Size-Dependent Generation of Reactive Oxygen Species","volume":"112","author":"Carlson","year":"2008","journal-title":"J. Phys. Chem. B"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1311","DOI":"10.1002\/jat.3315","article-title":"Silver nanoparticles induce pro-inflammatory gene expression and inflammasome activation in human monocytes","volume":"36","author":"Murphy","year":"2016","journal-title":"J. Appl. Toxicol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1016\/j.nano.2011.06.014","article-title":"Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles","volume":"8","author":"Thi","year":"2012","journal-title":"Nanomedicine"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Mahmoud, A., Moussa, S., El Backly, R., and El-Gendy, R. (2022). Investigating the residual effect of silver nanoparticles gel as an intra-canal medicament on dental pulp stromal cells. BMC Oral Health, 22.","DOI":"10.1186\/s12903-022-02542-2"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1002\/term.71","article-title":"In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages","volume":"2","author":"Zhang","year":"2008","journal-title":"J. Tissue Eng. Regen. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1891","DOI":"10.1089\/ten.tea.2009.0732","article-title":"A Feasibility of Useful Cell-Based Therapy by Bone Regeneration with Deciduous Tooth Stem Cells, Dental Pulp Stem Cells, or Bone-Marrow-Derived Mesenchymal Stem Cells for Clinical Study Using Tissue Engineering Technology","volume":"16","author":"Yamada","year":"2010","journal-title":"Tissue Eng. A"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.joen.2017.08.018","article-title":"Cytokine Expression of Stem Cells Originating from the Apical Complex and Coronal Pulp of Immature Teeth","volume":"44","author":"Joo","year":"2018","journal-title":"J. Endod."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9810","DOI":"10.1016\/j.biomaterials.2011.08.085","article-title":"The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles","volume":"32","author":"Park","year":"2011","journal-title":"Biomaterials"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1016\/j.joen.2013.11.008","article-title":"Effect of Dentin Conditioning with Intracanal Medicaments on Survival of Stem Cells of Apical Papilla","volume":"40","author":"Althumairy","year":"2014","journal-title":"J. Endod."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.1016\/j.joen.2012.06.018","article-title":"Direct Effect of Intracanal Medicaments on Survival of Stem Cells of the Apical Papilla","volume":"38","author":"Ruparel","year":"2012","journal-title":"J. Endod."},{"key":"ref_26","first-page":"1","article-title":"Comparison of Cytotoxic Effects of Silver Nanoparticles and Calcium Hydroxide on Human Gingival Fibroblasts: An In-vitro Study","volume":"5","author":"Saad","year":"2024","journal-title":"J. Dent. Health Oral Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1002\/jat.2792","article-title":"Silver nanoparticle-induced oxidative stress, genotoxicity and apoptosis in cultured cells and animal tissues","volume":"33","author":"Kim","year":"2013","journal-title":"J. Appl. Toxicol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1368","DOI":"10.1016\/j.joen.2012.06.036","article-title":"Effects of Calcium Hydroxide on Cytokine Expression in Endodontic Infections","volume":"38","author":"Tavares","year":"2012","journal-title":"J. Endod."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6858","DOI":"10.1016\/j.biomaterials.2012.06.016","article-title":"Inflammasome formation and IL-1\u03b2 release by human blood monocytes in response to silver nanoparticles","volume":"33","author":"Yang","year":"2012","journal-title":"Biomaterials"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1360","DOI":"10.1016\/j.joen.2008.08.020","article-title":"Effect of Calcium Hydroxide on Proinflammatory Cytokines and Neuropeptides","volume":"34","author":"Khan","year":"2008","journal-title":"J. Endod."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1016\/j.joen.2013.10.029","article-title":"Cellular and Molecular Tissue Response to Triple Antibiotic Intracanal Dressing","volume":"40","author":"Pereira","year":"2014","journal-title":"J. Endod."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/j.joen.2014.11.021","article-title":"Influence of Root Canal Disinfectants on Growth Factor Release from Dentin","volume":"41","author":"Galler","year":"2015","journal-title":"J. Endod."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1002\/jcb.1200","article-title":"Regulation of osteoclastogenesis and RANK expression by TGF-\u03b21","volume":"83","author":"Yan","year":"2001","journal-title":"J. Cell Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1949","DOI":"10.1016\/j.nano.2015.07.016","article-title":"Silver nanoparticles promote osteogenesis of mesenchymal stem cells and improve bone fracture healing in osteogenesis mechanism mouse model","volume":"11","author":"Zhang","year":"2015","journal-title":"Nanomedicine"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"34","DOI":"10.4103\/AMJM.AMJM_11_21","article-title":"An in-vitro Comparative Evaluation of Quantitative Release of Transforming Growth Factor \u03b2-1 from Dentin upon the Action of Endodontic Irrigants, Medicaments, Ultrasonic Activation, and Low-Level Laser Irradiation","volume":"17","author":"Akhila","year":"2021","journal-title":"Amrita J. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1046\/j.1365-2591.2000.00326.x","article-title":"TGF-\u03b21 alone and in combination with calcium hydroxide is synergistic to TGF-\u03b21 production by osteoblasts in vitro","volume":"33","author":"Jaunberzins","year":"2000","journal-title":"Int. Endod. J."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Pan, H., Yang, Y., Xu, H., Jin, A., Huang, X., Gao, X., Sun, S., Liu, Y., Liu, J., and Lu, T. (2023). The odontoblastic differentiation of dental mesenchymal stem cells: Molecular regulation mechanism and related genetic syndromes. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.1174579"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2025","DOI":"10.1016\/j.joen.2017.06.018","article-title":"Revascularization-associated Intracanal Calcification: Assessment of Prevalence and Contributing Factors","volume":"43","author":"Song","year":"2017","journal-title":"J. Endod."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1580842","DOI":"10.1155\/2022\/1580842","article-title":"The Four Pillars for Successful Regenerative Therapy in Endodontics: Stem Cells, Biomaterials, Growth Factors, and Their Synergistic Interactions","volume":"2022","author":"Brizuela","year":"2022","journal-title":"Stem Cells Int."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1911","DOI":"10.1089\/ten.tea.2010.0615","article-title":"Complete Pulp Regeneration After Pulpectomy by Transplantation of CD105 + Stem Cells with Stromal Cell-Derived Factor-1","volume":"17","author":"Iohara","year":"2011","journal-title":"Tissue Eng A"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1089\/ten.tea.2014.0154","article-title":"The Interplay of Dental Pulp Stem Cells and Endothelial Cells in an Injectable Peptide Hydrogel on Angiogenesis and Pulp Regeneration In Vivo","volume":"21","author":"Dissanayaka","year":"2015","journal-title":"Tissue Eng. A"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1016\/j.joen.2011.12.024","article-title":"Coculture of Dental Pulp Stem Cells with Endothelial Cells Enhances Osteo-\/Odontogenic and Angiogenic Potential In Vitro","volume":"38","author":"Dissanayaka","year":"2012","journal-title":"J. Endod."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.actbio.2016.02.040","article-title":"Pulp regeneration in a full-length human tooth root using a hierarchical nanofibrous microsphere system","volume":"35","author":"Li","year":"2016","journal-title":"Acta Biomater."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1070","DOI":"10.1016\/j.joen.2016.04.023","article-title":"Characteristics and Effects on Dental Pulp Cells of a Polycaprolactone\/Submicron Bioactive Glass Composite Scaffold","volume":"42","author":"Wang","year":"2016","journal-title":"J. Endod."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1137","DOI":"10.1177\/0022034518772260","article-title":"Pulp Regeneration by 3-dimensional Dental Pulp Stem Cell Constructs","volume":"97","author":"Itoh","year":"2018","journal-title":"J. Dent. Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1460","DOI":"10.1177\/0022034518785124","article-title":"Decellularized Swine Dental Pulp Tissue for Regenerative Root Canal Therapy","volume":"97","author":"Alqahtani","year":"2018","journal-title":"J. Dent. Res."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Aydin, S., and \u015eahin, F. (2019). Stem Cells Derived from Dental Tissues. Stem Cells Derived from Dental Tissues, Springer.","DOI":"10.1007\/5584_2018_333"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"709","DOI":"10.1016\/j.archoralbio.2010.12.008","article-title":"Comparative analysis of in vitro osteo\/odontogenic differentiation potential of human dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP)","volume":"56","author":"Bakopoulou","year":"2011","journal-title":"Arch. Oral. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"538","DOI":"10.1016\/j.joen.2013.09.015","article-title":"Histopathological Condition of the Remaining Tissues after Endodontic Infection of Rat Immature Teeth","volume":"40","author":"Duarte","year":"2014","journal-title":"J. Endod."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Liu, Q., Gao, Y., and He, J. (2023). Stem Cells from the Apical Papilla (SCAPs): Past, Present, Prospects, and Challenges. Biomedicines, 11.","DOI":"10.3390\/biomedicines11072047"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1111\/iej.13701","article-title":"A critical analysis of research methods and experimental models to study biocompatibility of endodontic materials","volume":"55","author":"Hosseinpour","year":"2022","journal-title":"Int. Endod. J."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1111\/j.1365-2591.1980.tb00671.x","article-title":"A comparison of in vivo and in vitro methods for toxicity testing of dental materials","volume":"13","author":"Mjor","year":"1980","journal-title":"Int. Endod. J."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"S6","DOI":"10.1016\/0300-5712(94)90032-9","article-title":"Use of cell cultures for toxicity testing of dental materials\u2014Advantages and limitations","volume":"22","author":"Schmalz","year":"1994","journal-title":"J. Dent."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1046\/j.1365-2591.1999.00232.x","article-title":"Calcium hydroxide pastes: Classification and clinical indications","volume":"32","author":"Fava","year":"1999","journal-title":"Int. Endod. J."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/21\/10604\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T07:31:17Z","timestamp":1761895877000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/21\/10604"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,31]]},"references-count":54,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["ijms262110604"],"URL":"https:\/\/doi.org\/10.3390\/ijms262110604","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,31]]}}}