Effect of Bacterial Biofilm on the Osteogenic Differentiation of Stem Cells of Apical Papilla Lakshmi Vishwanat, DDS,* Rose Duong, BS,* Koyo Takimoto, DDS,* Linda Phillips, MS, † Claudia O. Espitia, MS,* Anibal Diogenes, DDS, MS, PhD,* Shivani B. Ruparel, PhD,* David Kolodrubetz, PhD, † and Nikita B. Ruparel, MS, DDS, PhD* Abstract Introduction: Although clinical success in regenera- tive endodontics is substantially high, histological success is limited to finding bone/cementum-like tis- sue instead of dentin within the canal space. The aims of this study were to investigate (1) the effect of bacterial biofilm on osteogenic gene expression in stem cells of the apical papilla (SCAP) and (2) the effect of bacterial antigens on the functional differen- tiation of SCAP into a mineralizing phenotype. Methods: Using an ex vivo organotypic root canal model and an American Association of Endontists- recommended regenerative endodontic procedures, we evaluated SCAP differentiation in the presence and absence of an Enterococcus faecalis biofilm. Gene expression analysis for dentinogenic and osteo- blastic markers was performed with real-time poly- merase chain reaction. The effect of E. faecalis antigens on SCAP differentiation into mineralizing cells in vitro was evaluated with 2 functional assays: Alizarin Red and alkaline phosphatase activity assays. Results: After regenerative endodontic procedures, residual bacteria continued to sustain within the root canal system. SCAP in the presence of E. faeca- lis biofilm significantly downregulated dentinogenic genes such as dentin sialophosphoprotein and upre- gulated osteoblastic genes such as bone sialoprotein, osteocalcin, distal-less homeobox 5, and runt-related transcription factor 2. E. faecalis antigens signifi- cantly inhibited SCAP differentiation into a mineral- izing phenotype when alizarin red staining and alkaline phosphatase assays were used in vitro. Conclusions: Current disinfection protocols were ineffective in eliminating bacteria from root tips and the levels of the residual bacterial biofilm, and its by- products, were able to significantly alter osteogenic- differentiation of SCAP. (J Endod 2017;-:1–7) Key Words Bacterial antigens, biofilm, dentinogenic, osteoblastic, regenerative endodontic proced- ures, stem cells of apical papilla C linical success with regenerative endodon- tic procedures (REPs), based on case reports, case series, and random- ized clinical trials, appears to be substantially high. Clinical success has been defined as healing of apical periodontitis, gain in root width and length for an immature tooth, and possibly restoration of nerve function (1). These criteria for success address patient-centered as well as clinician-centered outcomes (1). Success as measured by scientist-centered outcomes, such as histology, however, is limited to noninfected cases. Histologic success would be described as regenerating lost or damaged cells of the pulp- dentin complex as well as regeneration of tissues of the pulp-dentin complex, such as fibrous connective tissue, vasculature, innervation, and dentin-like tissue. Currently, there are few clinical studies that have examined the histology of pulp and dentin tissue after REPs (2–4), although REPs performed in teeth with vital pulps, in either human (5) or animal studies (6–8), show promising results. For example, Shimizu et al (5) reported that an REP-treated tooth with a diagnosis of irreversible pul- pitis exhibited flattened odontoblast-like cells lining the predentin. In contrast, the avail- able literature implicates bacteria in altered histologic outcomes. Nearly all histologic analyses of soft tissues after REP demonstrate regeneration of fibrous connective tissue as well as vasculature within the root canal space (2–4), but the majority of hard tissue histologic analyses, barring one report (5), indicate osteoid- or bone-like and/or ce- mentoid- or cementum-like tissue formation in the regenerated section of these teeth (2–4), thus, dentin formation commonly is not observed. Importantly, the majority of these cases had a preoperative diagnosis of pulpal necrosis with either symptomatic apical periodontitis or acute/chronic apical abscess. These diagnoses presumably are associated with a long-standing infection and biofilm in the canal spaces of these teeth. In support of this, several studies in animals have demonstrated that in- fected necrotic pulps treated with REP resulted in bone and cementum-like hard tissue deposition instead of dentin (9–11). From the *Department of Endodontics and † Microbiology, Immunology & Molecular Genetics, University of Texas Health Science Center San Antonio, San Antonio, Texas. Address requests for reprints to Nikita B. Ruparel, Assistant Professor, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: ruparel@uthscsa.edu 0099-2399/$ - see front matter Copyright ª 2017 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2017.01.023 Significance The present study demonstrates that residual bio- film promotes osteoblastic versus dentinogenic gene expression of stem cells of apical papilla and that bacterial antigens significantly inhibit osteogenic differentiation of stem cells of apical papilla. Adequate detoxification of canal system is therefore warranted in regenerative endodontic procedures. Regenerative Endodontics JOE — Volume -, Number -, - 2017 Bacterial Biofilm on the Osteogenic Differentiation of SCAP 1