A Critical Review of In Vitro Methodologies for Studying Inflammatory Responses in Human Dental Pulp Cell Cultures.

Background: The pulp responds to disease and trauma through inflammation which modulates the tooth's ability to instigate healing mechanisms. In vitro human dental pulp cell (HDPC) culture models are widely used to study the tooth's response to pro-inflammatory stimuli.

Objectives: The purpose of this review is to generate a structured appraisal of in vitro research methodologies to study HDPC cultures in a state of inflammation.

Method: In this narrative review the Scopus, PubMed and Google Scholar databases were searched to identify studies reporting the use of HDPC cultures in vitro in inflammation-associated research until December 2024. The dataset initially identified 642 publications, which were manually screened to identify 246 relevant studies for inclusion and methodologically mined for information on: (i) study purpose, (ii) source and characterisation of cells, (iii) pro-inflammatory stimuli used and (iv) assays and markers used to characterise the inflammatory response. The collected data underpinned this review.

Results: Most published studies aimed to characterise HDPC responses to a range of pro-inflammatory stimulants which included bacterial components (lipopolysaccharides [LPS] and lipoteichoic acids [LTA]), cytokines and biomaterials. These stimulations were studied: to characterise their effect in the development of new scaffolds and dental (bio)materials and to mimic the in vivo environment. Various tooth sources were used to establish HDPC cultures, and predominantly, cells were isolated using the pulp tissue explant technique. The most frequently used stimulant and concentrations were LPS at 1 and 10 μg/mL. The time for cell stimulation prior to inflammatory response assay varied from 15 min to 10 days. Inflammatory assessments were performed using quantitative polymerase chain reaction (qPCR), Western Blotting, enzyme-linked immunosorbent assay (ELISA) and high-throughput assays targeting archetypical cytokines and dentinogenic and mineralisation-associated molecules.

Discussion: This review highlights the broad range of experimental conditions used to study HDPC inflammatory responses in vitro and combines these data to identify a framework for a consensus and a more uniform experimental approach.

Conclusion: Standardisation of in vitro experimental conditions to evaluate the pulps response to inflammatory stimuli would enhance research rigour and improve clinical translation to ultimately inform patient treatment and outcomes.