{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,25]],"date-time":"2026-03-25T23:10:10Z","timestamp":1774480210410,"version":"3.50.1"},"reference-count":36,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T00:00:00Z","timestamp":1773187200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002322","name":"CAPES","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100002322","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100004586","name":"FAPERJ","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100004586","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003593","name":"CNPq","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100003593","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>Hydroxyapatite-based ceramics are widely used in dental bioengineering, yet the reliable assessment of endotoxin activity in solid porous materials remains challenging. This study evaluated the applicability of a whole blood Monocyte Activation Test (MAT) to a hydroxyapatite ceramic relevant to dental applications. Two endotoxin association strategies (immersion and dropwise) were compared, followed by nonlinear modelling of cytokine dose\u2013response curves using four-parameter logistic (4PL) regression and spike-recovery analysis to assess potential material interference. Immersion-based spiking produced reproducible, concentration-dependent cytokine responses, whereas dropwise application resulted in minimal functional recovery. IL-1\u03b2, IL-6, and TNF-\u03b1 displayed sigmoidal dose\u2013response profiles with high goodness-of-fit values (R2 \u2265 0.93). Spike recovery remained within the 50\u2013200% acceptance range for most concentrations, with IL-6 showing the most consistent analytical performance. TNF-\u03b1 exhibited signal saturation at higher endotoxin levels, limiting its dynamic range. Multiplex cytokine profiling confirmed that classical MAT readouts were among the most strongly induced mediators and that hydroxyapatite did not trigger baseline inflammatory activation. These findings demonstrate that whole blood MAT can be reliably applied to hydroxyapatite-based dental ceramics when immersion-based endotoxin association and material-specific methodological optimization are employed.<\/jats:p>","DOI":"10.3390\/bioengineering13030319","type":"journal-article","created":{"date-parts":[[2026,3,11]],"date-time":"2026-03-11T09:00:37Z","timestamp":1773219637000},"page":"319","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Applicability of Whole Blood Monocyte Activation Test for Endotoxin Activity Assessment in Hydroxyapatite-Based Ceramics"],"prefix":"10.3390","volume":"13","author":[{"given":"Janaina","family":"Spoladore","sequence":"first","affiliation":[{"name":"Post-Graduation Program in Science and Biotechnology, Institute of Biology, Fluminense Federal University, Niteroi 24210-201, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carolina Barbara","family":"Nogueira de Oliveira","sequence":"additional","affiliation":[{"name":"Brazilian Center for Validation of Alternative Methods (BraCVAM), Instituto de Ci\u00eancia e Tecnologia em Biomodelos (ICTB), FIOCRUZ, Rio de Janeiro 21040-360, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joice","family":"Correa da Silva","sequence":"additional","affiliation":[{"name":"Post-Graduation Program in Science and Biotechnology, Institute of Biology, Fluminense Federal University, Niteroi 24210-201, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena","family":"Mavropoulos Tude","sequence":"additional","affiliation":[{"name":"Brazilian Center for Physics Research, Rio de Janeiro 22290-180, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5775-0222","authenticated-orcid":false,"given":"Carlos Fernando","family":"Mour\u00e3o","sequence":"additional","affiliation":[{"name":"Department of Clinical and Translational Research, Tufts University School of Dental Medicine, Boston, MA 02111, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0016-4809","authenticated-orcid":false,"given":"Gutemberg Gomes","family":"Alves","sequence":"additional","affiliation":[{"name":"Clinical Research Unit, Ant\u00f4nio Pedro University Hospital, Fluminense Federal University, Niteroi 24033-900, Brazil"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,11]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Biomaterials for bone tissue engineering: Achievements to date and future directions","volume":"20","author":"Garimella","year":"2024","journal-title":"Biomed. Mater."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1080\/17460751.2024.2343555","article-title":"Bioactive ceramic-based materials: Beneficial properties and potential applications in dental repair and regeneration","volume":"19","author":"Wang","year":"2024","journal-title":"Regen. Med."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Krbec, D., and Krukowski, S. (2025). Biomedical applications of polymer-coated hydroxyapatite materials. Prospect. Pharm. Sci., 1\u201312.","DOI":"10.56782\/pps.681"},{"key":"ref_4","unstructured":"(2024). Biological Evaluation of Medical Devices. Part 1. Evaluation and Testing Within a Risk Management Process (Standard No. BS EN ISO 10993-1)."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1089\/ten.teb.2012.0636","article-title":"Endotoxins-the invisible companion in biomaterials research","volume":"19","author":"Lieder","year":"2013","journal-title":"Tissue Eng. Part B Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"153","DOI":"10.2345\/0899-8205-57.4.153","article-title":"Affinity and Inactivation of Bacterial Endotoxins for Medical Device Materials","volume":"57","author":"Kimble","year":"2023","journal-title":"Biomed. Instrum. Technol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"7001","DOI":"10.12659\/MSM.910039","article-title":"Endotoxin Contributes to Artificial Loosening of Prostheses Induced by Titanium Particles","volume":"24","author":"Wang","year":"2018","journal-title":"Med. Sci. Monit."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Tamura, H., Reich, J., and Nagaoka, I. (2021). Outstanding Contributions of LAL Technology to Pharmaceutical and Medical Science: Review of Methods, Progress, Challenges, and Future Perspectives in Early Detection and Management of Bacterial Infections and Invasive Fungal Diseases. Biomedicines, 9.","DOI":"10.3390\/biomedicines9050536"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"105160","DOI":"10.1016\/j.tiv.2021.105160","article-title":"Standardized pyrogen testing of medical products with the bacterial endotoxin test (BET) as a substitute for rabbit Pyrogen testing (RPT): A scoping review","volume":"74","author":"Spoladore","year":"2021","journal-title":"Toxicol. In Vitro"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1177\/1753425913492833","article-title":"Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay","volume":"20","author":"Kucki","year":"2014","journal-title":"Innate Immun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1177\/17534259221097948","article-title":"The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens","volume":"28","author":"Solati","year":"2022","journal-title":"Innate Immun."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Gimenes, I., Spoladore, J., Paranhos, B.A., Romasco, T., Di Pietro, N., Piattelli, A., Mourao, C.F., and Gomes Alves, G. (2024). Assessment of Pyrogenic Response of Medical Devices and Biomaterials by the Monocyte Activation Test (MAT): A Systematic Review. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25147844"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"453","DOI":"10.14573\/altex.1709221","article-title":"Material-mediated pyrogens in medical devices: Applicability of the in vitro Monocyte Activation Test","volume":"35","author":"Borton","year":"2018","journal-title":"Altex"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2739","DOI":"10.1016\/S0142-9612(03)00089-9","article-title":"Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro","volume":"24","author":"Laquerriere","year":"2003","journal-title":"Biomaterials"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2922","DOI":"10.1038\/s41598-017-03086-0","article-title":"The shape and size of hydroxyapatite particles dictate inflammatory responses following implantation","volume":"7","author":"Lebre","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_16","first-page":"351","article-title":"Effects of albumin adsorption on cell adhesion in hydroxyapatite modified surfaces","volume":"631","author":"Mavropoulos","year":"2015","journal-title":"Key Eng. Mater."},{"key":"ref_17","unstructured":"(2024). Amd 1 Biological Evaluation of Medical Devices. Part 12: Sample Preparation and Reference Materials (Standard No. BS EN ISO 10993-12:2021)."},{"key":"ref_18","first-page":"317","article-title":"Quality-control analytical methods: Pharmacopeial bacterial endotoxins test chapters","volume":"14","author":"Dawson","year":"2010","journal-title":"Int. J. Pharm. Compd."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"100206","DOI":"10.1016\/j.crtox.2024.100206","article-title":"Fit for purpose testing and independent GMP validation of the monocyte activation test","volume":"8","author":"Daniels","year":"2025","journal-title":"Curr. Res. Toxicol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1781","DOI":"10.1039\/C9TB02710F","article-title":"Substituted hydroxyapatite coatings of bone implants","volume":"8","author":"Arcos","year":"2020","journal-title":"J. Mater. Chem. B"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1042","DOI":"10.1016\/j.addr.2011.06.012","article-title":"Innate immunity activation on biomaterial surfaces: A mechanistic model and coping strategies","volume":"63","author":"Ekdahl","year":"2011","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1111\/j.1600-051X.2012.01929.x","article-title":"Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: A preliminary study","volume":"39","author":"Harder","year":"2012","journal-title":"J. Clin. Periodontol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1016\/j.biomaterials.2006.11.016","article-title":"An in vitro pyrogen safety test for immune-stimulating components on surfaces","volume":"28","author":"Hasiwa","year":"2007","journal-title":"Biomaterials"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"276","DOI":"10.1002\/jbm.a.30922","article-title":"In vitro pyrogen test--A new test method for solid medical devices","volume":"80","author":"Mazzotti","year":"2007","journal-title":"J. Biomed. Mater. Res. A"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1177\/026119291604400305","article-title":"More than 70 years of pyrogen detection: Current state and future perspectives","volume":"44","author":"Fennrich","year":"2016","journal-title":"Altern. Lab. Anim."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1065","DOI":"10.1007\/s10856-013-5136-6","article-title":"Highly sensitive pyrogen detection on medical devices by the monocyte activation test","volume":"25","author":"Stang","year":"2014","journal-title":"J. Mater. Sci. Mater. Med."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Yang, Q., Li, Y., Tuohuti, P., Qin, Z., Zhang, Z., Zhao, W., and Su, B. (2021). Advances in the Development of Biomaterials for Endotoxin Adsorption in Sepsis. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.699418"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1170","DOI":"10.1016\/j.jpba.2011.04.005","article-title":"Detection of pyrogenicity on medical grade polymer materials using rabbit pyrogen, LAL and ELISA method","volume":"55","author":"Mohanan","year":"2011","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/0022-1759(95)00184-C","article-title":"Variable estimates of cytokine levels produced by commercial ELISA kits: Results using international cytokine standards","volume":"186","author":"Ledur","year":"1995","journal-title":"J. Immunol. Methods"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1177\/1753425918777596","article-title":"Analysis of IL-6 and IL-1beta release in cryopreserved pooled human whole blood stimulated with endotoxin","volume":"24","author":"He","year":"2018","journal-title":"Innate Immun."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Assoratgoon, I., Nowwarote, N., Phothichailert, S., Dissanayaka, W.L., Samaranayake, L.P., and Osathanon, T. (2025). Unravelling the role of interleukin-6 in regulating dental stem cell behaviour: A scoping review. BMC Oral Health, 25.","DOI":"10.1186\/s12903-025-06097-w"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e35","DOI":"10.1111\/clr.12326","article-title":"Comparison of pro-inflammatory cytokines and bone metabolism mediators around titanium and zirconia dental implant abutments following a minimum of 6 months of clinical function","volume":"26","author":"Barwacz","year":"2015","journal-title":"Clin. Oral Implants Res."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Tylutka, A., Walas, L., and Zembron-Lacny, A. (2024). Level of IL-6, TNF, and IL-1beta and age-related diseases: A systematic review and meta-analysis. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1330386"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Fadeeva, I.S., Teterina, A.Y., Smirnov, I.V., Minaychev, V.V., Shlykov, M.A., Kobyakova, M.I., Smirnova, P.V., Senotov, A.S., Zvyagina, A.I., and Palikov, V.A. (2026). Differential Immune Response to Hydroxyapatite Precursors Under Inflammatory Pressure: In Vitro and In Vivo Studies. Cells, 15.","DOI":"10.3390\/cells15020101"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Zhao, R., Yang, R., Cooper, P.R., Khurshid, Z., Shavandi, A., and Ratnayake, J. (2021). Bone Grafts and Substitutes in Dentistry: A Review of Current Trends and Developments. Molecules, 26.","DOI":"10.3390\/molecules26103007"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.3390\/jfb6041099","article-title":"Fabrication, Properties and Applications of Dense Hydroxyapatite: A Review","volume":"6","author":"Prakasam","year":"2015","journal-title":"J. Funct. Biomater."}],"container-title":["Bioengineering"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-5354\/13\/3\/319\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,13]],"date-time":"2026-03-13T05:21:02Z","timestamp":1773379262000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-5354\/13\/3\/319"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,11]]},"references-count":36,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["bioengineering13030319"],"URL":"https:\/\/doi.org\/10.3390\/bioengineering13030319","relation":{},"ISSN":["2306-5354"],"issn-type":[{"value":"2306-5354","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,11]]}}}