3D-Printed and Bioprinted Scaffolds in Regenerative Endodontics: A Systematic Review

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This systematic review synthesizes how 3D-printed/bioprinted scaffolds and bioinks are being engineered for regenerative endodontics and identifies the most promising material strategies to enhance cell viability, odontogenic differentiation, mineralization, and (when assessed) antimicrobial activity. It offers a practical guide to selecting printable biomaterials (e.g., calcium silicate-polymer composites, dentin-matrix/alginate hydrogels, and functionalized PLA) and designing patient-tailored scaffolds that pair pro-regenerative cues with controlled antimicrobial function to improve outcome predictability.

Abstract

Introduction: Three-dimensional (3D) bioprinting is a promising approach for endodontic tissue engineering, enabling scaffolds with controlled architecture and bioactivity to support pulp regeneration. Objectives: This systematic review assessed the following: “What 3D bioprinting applications are reported in endodontics-related studies?” Materials and Methods: Following PRISMA 2020 guidelines, PubMed/MEDLINE, Scopus, Embase, Cochrane Library, Web of Science, SciELO, LILACS, and Google Scholar were searched up to January 2026 with no date or language limits. Two reviewers independently screened studies; risk of bias in in vitro studies was assessed with the QUIN tool. As only one study reported complete antimicrobial outcomes, an intra-study quantitative comparison (MD, 95% CI) of inhibition halos was performed (not a meta-analysis). Results: From 518 records, nine studies were included. Outcomes mainly addressed physicochemical properties (n = 9), cell viability (n = 7), biocompatibility (n = 5), and cell differentiation (n = 5); antimicrobial activity was evaluated in two studies. Most used hDPSCs and extrusion-based printing, testing calcium silicate composites, alginate hydrogels, functionalized PCL, and modified PLA. Modified PLA scaffolds showed greater antimicrobial activity, strongest with naringin and nHA formulations. Overall risk of bias was moderate (58.33%), largely due to limited reporting of randomization, blinding, and sampling. Conclusion: 3D-bioprinted scaffolds/bioinks generally improved cellular responses and bioactivity, especially with MTA, Biodentine, nHA, or naringin; antimicrobial effects were most evident in functionalized PLA (PLA/NAR and PLA/nHA/NAR).

1. Introduction

Caries, pulpitis, and apical periodontitis are highly prevalent conditions that reduce quality of life and impose substantial healthcare and economic burdens [1,2]. These impacts are particularly acute in immature permanent teeth, where pulp necrosis caused by caries, trauma, or developmental anomalies arrests root maturation, leaving open apices and thin dentinal walls that complicate conventional endodontic treatment [3,4,5]. Regenerative endodontic procedures (REPs) were introduced to overcome these limitations by aiming to restore a functional dentin–pulp complex and promote continued root development rather than merely disinfecting and filling the canal space [4,6]. However, despite encouraging clinical outcomes, REPs remain variably predictable, with failures often linked to persistent infection and/or insufficient tissue regeneration [7,8,9]. A central debate in the field concerns whether current protocols reliably regenerate pulp-like tissue or instead produce repair tissues of variable composition and how intensifying antimicrobial strategies can compromise cell survival and regenerative signaling.

The biological rationale for REPs draws on tissue engineering concepts, combining cells, scaffolds, biomaterials, and signaling cues to regenerate damaged tissues [10,11]. Human dental pulp stem cells (hDPSCs) and related dental progenitors can differentiate toward odontoblast-like lineages and contribute to dentin–pulp complex formation in vivo, supporting their use in regenerative strategies [12,13]. In this context, scaffolds act as three-dimensional templates that help organize cells and guide tissue formation; to be effective, they must be biocompatible, appropriately porous and interconnected, biodegradable, and mechanically suitable for the target environment [14,15].

A wide range of candidate scaffold materials has been explored. Hydrogels, such as fibrin, agarose, polyethylene glycol (PEG) derivatives, alginate, and gelatin, are attractive because they can mimic soft extracellular matrices and support cell encapsulation [12,16,17]. Hybrid alginate–gelatin (Alg-Gel) systems can improve printability and microenvironmental cues for cell adhesion and proliferation [12,18,19]. Biomimetic bioinks incorporating dentin extracellular matrix (e.g., Alg-Dent) aim to combine the biological functionality of dentin-derived components, which are rich in collagenous and non-collagenous factors, with the printability and processability of alginate [20,21]. In parallel, calcium silicate-based materials remain central to regenerative endodontics. Mineral trioxide aggregate (MTA) is widely used for its sealing ability and bioactivity, including ion release and stimulation of mineralization-related pathways [22,23]. Biodentine has been reported to show improved mechanical performance compared with MTA and to promote odontogenic/osteogenic responses in multiple cell types [24,25], yet practical limitations (e.g., handling and setting characteristics) and mechanical constraints have motivated new formulations and polymer composites [2,26].

Additive manufacturing (3D printing) extends these strategies by enabling scaffolds with precisely controlled architectures and patient-specific geometries [2,22,27]. Extrusion-based bioprinting and fused deposition modeling (FDM), among other approaches, allow the layer-by-layer fabrication of constructs from polymers, ceramics, and composites, potentially integrating cells and bioactive agents in spatially defined patterns [27,28]. Synthetic polymers such as poly-ε-caprolactone (PCL) and polylactic acid (PLA) are commonly used due to biocompatibility, biodegradability, and favorable mechanics [22,29,30]. Nevertheless, their intrinsic hydrophobicity and limited bioactivity can reduce cell attachment and instructive signaling, driving functionalization with bioceramics and bioactive molecules (e.g., MTA, nanohydroxyapatite, naringin, leptin, bioactive glasses, and ion-doped nanoparticles) to enhance mineralization, modulate inflammation, and introduce antimicrobial effects [9,31]. Complementary approaches include gelatin-based hydrogels and GelMA systems loaded with antiseptics, antibiotics, or quaternary ammonium compounds as controlled-release platforms against endodontic biofilms, including Enterococcus faecalis [31,32]. Here, another key controversy emerges as antimicrobial potency must be balanced against cytotoxicity and the preservation of growth factors and cell function within a regenerating canal space [9,33].

Persistent infection remains one of the main barriers to REP success [9,34]. Enterococcus faecalis is frequently implicated in endodontic failure because it tolerates harsh environments, penetrates dentinal tubules, and forms resilient biofilms [7]. Conventional irrigation and intracanal medicaments may require concentrations that harm host cells or diminish pro-regenerative signals, potentially undermining the very biology that REPs depend on [9,33]. Against this backdrop, 3D printing and bioprinting are particularly compelling because they can combine tailored architecture, bioactive cue presentation, and controlled antimicrobial release within a scaffold designed to support hDPSC survival and differentiation. Yet, the field remains fragmented, with substantial heterogeneity in materials, printing methods, cell sources, models, and reported outcomes, limiting cross-study comparison and clinical translation [4,35].

To address this need, the present systematic review critically synthesizes preclinical evidence on 3D-printed scaffolds and bioprinted bioinks applied to endodontics, guided by the PICOS question: “What are the applications of 3D bioprinting described in the scientific literature in studies related to endodontics?”.

2. Materials and Methods

2.1. Protocol and Registration

The materials and methods were based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines (Table S1) [36,37]. A research protocol was registered in the Open Science Framework (OSF), registration DOI 10.17605/OSF.IO/N23G5.

2.2. Research Question and Eligibility Framework (PICOS)

The review was designed to answer the following question: “What are the applications of 3D bioprinting described in the scientific literature in studies related to endodontics?”. The PICOS framework was defined as follows: the population (P) comprised experimental studies related to endodontics (in vitro, ex vivo, in vivo and clinical); the intervention (I) involved the application of 3D bioprinting/3D printing for biologically relevant endodontic purposes, including different techniques, biomaterials, and cell sources; the comparison (C) was not applicable; the outcomes (O) included applications and methodological approaches, biomaterials/bioinks, scaffold constructs, cell-related outcomes (e.g., viability and differentiation), mineralization/bioactivity, dentin–pulp complex regeneration, physicochemical characterization, and antimicrobial testing when reported; and the study design (S) included original research articles published in scientific journals.

2.3. Information Sources and Search Strategy

Electronic searches were performed in PubMed/MEDLINE, Scopus, Embase, Cochrane Library, Web of Science, SciELO, and LILACS from inception to 30 January 2026. Gray literature was searched using Google Scholar, and the reference lists of included studies were manually screened to identify additional eligible records.

The search strategy combined keywords and Medical Subject Headings (MeSH) using the Boolean operators AND and OR. The full strategies for all databases are provided in Table S2. No restrictions were applied regarding publication year or language.

2.4. Eligibility Criteria

Eligibility criteria were predefined to ensure consistency during study selection and to capture experimental evidence specifically addressing biologically relevant applications of 3D printing/bioprinting in endodontics. Studies were included or excluded according to the criteria summarized in

Table 1. Inclusion and exclusion criteria applied to determine study eligibility for the review.

Domain	Inclusion Criteria	Exclusion Criteria
Study type	Original experimental studies (in vitro, ex vivo, in vivo or clinical)	Narrative/systematic/scoping reviews; editorials; letters; conference abstracts; book chapters
Scope/Relevance	Studies addressing endodontic procedures and/or dental pulp regeneration	Applications exclusively in other dental fields without endodontic/pulp relevance
Technology of interest	3D bioprinting involving bioprinted constructs and/or bioinks, including cell-laden or biologically functionalized inks intended for pulp/endodontic applications	Non-biological 3D printing applications
Experimental model	Cell-based and/or bioactive strategies relevant to pulp/endodontic tissue engineering	Studies not relevant to endodontics or without relevance to pulpal/periapical tissues
Outcomes reported	At least one relevant outcome: cell viability, proliferation, differentiation, ECM/mineralization, vascularization proxies, biocompatibility, printability linked to biological function	Outcomes unrelated to pulp/endodontic goals with no pulp/endodontic component
Language/Timeframe	No restrictions on language or publication date	Duplicates or multiple reports of the same dataset
Availability	Full text accessible	Full text unavailable after reasonable efforts

.

2.5. Study Selection

Study selection was performed independently by two reviewers (H.G.S.C. and V.C.M.D.) in two stages. Duplicates were identified and removed using EndNote (v21.0; Clarivate Analytics, Philadelphia, PA, USA). Title/abstract screening was conducted using Rayyan (Rayyan, Qatar Computing Research Institute, Qatar Foundation). Records meeting eligibility criteria were included; when abstracts provided insufficient information, full texts were retrieved.

Full-text eligibility was assessed independently by the reviewers. Disagreements were resolved by discussion; when necessary, a third reviewer (J.M.M.d.S.) adjudicated. Inter-reviewer agreement was quantified using Cohen’s kappa [37,38].

2.6. Data Extraction and Synthesis

Tables for data extraction were designed according to the following items. Table 2 summarizes study-level characteristics, including authors/year, study design, objective, experimental model, bioink/scaffold composition, printing approach, endodontic focus, and main conclusions. Table 3 compiles outcome- and methods-related variables, including cell type, viability assays, differentiation markers, mineralization/bioactivity outcomes, physicochemical properties, characterization methods, and antimicrobial testing.

2.7. Quantitative Synthesis (Intra-Study Analysis)

A quantitative intra-study analysis was performed using data from Dawood and Mahdee [39] to compute mean differences (MD) and 95% confidence intervals for inhibition halos (mm) of PLA/Nano-Hydroxyapatite (nHA), PLA/Naringin (NAR), and PLA/nHA/NAR relative to PLA. As this analysis derived from a single study, it does not constitute a between-study meta-analysis; it just complements the narrative synthesis.

2.8. Risk of Bias Assessment

Risk of bias was assessed using the QUIN tool for in vitro dental research [40,41]. QUIN includes 12 domains: (1) stated aims/objectives; (2) sample size calculation/justification; (3) sampling technique with inclusion/exclusion criteria; (4) details of comparison/control group; (5) methodological detail/standardization; (6) operator details; (7) randomization; (8) outcome measurement method; (9) assessor details and reliability; (10) blinding; (11) statistical analysis; and (12) results presentation.

Each item was scored as 2 (adequately specified), 1 (inadequately specified), or 0 (not specified). Not applicable (NA) items were excluded from the denominator. The QUIN percentage was calculated as QUIN % = (sum of points × 100)/[2 × number of applicable items], classifying studies as low (>70%), moderate (50–70%), or high (<50%) risk of bias. Two reviewers (H.G.d.S.C. and A.C.-F.) assessed risk independently; disagreements were resolved by consensus or adjudication.

3. Results

3.1. Study Selection

The selection process is presented in Figure 1. In total, 518 records were screened. After title/abstract screening (Step 1), 10 studies were assessed in full (Step 2). One study was excluded, with the reason reported in Figure 1 [42]. Ultimately, nine studies met eligibility criteria and were included in data tabulation [2,9,11,12,23,33,35,39,43].

Inter-reviewer kappa values indicated near-perfect agreement: 0.898 for PubMed/MEDLINE, 0.926 for Scopus, 0.945 for Embase, and 1.000 for Cochrane Library, Web of Science, LILACS, and Google Scholar [37]. Manual reference-list screening yielded no additional records.

3.2. Characteristics of Included Studies

Key study characteristics are summarized in Table 2 and Table 3. The nine included studies investigated 3D-printed scaffolds and/or bioprinted bioinks relevant to endodontics [2,9,11,12,23,33,35,39,43]. Across studies, outcomes most frequently assessed were physicochemical characteristics (n = 9), cell viability (n = 7), biocompatibility (n = 5), differentiation potential (n = 5), and antimicrobial activity (n = 2).

The included studies were published between 2017 and 2025, reflecting the relatively recent emergence of 3D printing and bioprinting approaches in regenerative endodontics. A temporal trend was observed, in which earlier studies primarily focused on scaffold fabrication and physicochemical characterization, often using acellular models or simplified systems. In contrast, more recent studies increasingly explored complex 3D-printed and bioprinted constructs, incorporating bioactive materials, functionalization strategies, and advanced biological models, indicating a shift toward more biologically relevant and multifunctional scaffold designs.

Most studies used hDPSCs as the primary cell model [2,9,12,23,43]. One study [9] employed stem cells from the apical papilla (SCAPs), while others used acellular printed inserts to evaluate release/diffusion kinetics [35] or focused on antibacterial performance of acellular scaffolds [43]. Biomaterials included MTA/PCL, Biodentine (BD)/PCL, Alg-Gel hydrogels, Alg-Dent formulations, aminated PCL loaded with leptin, fibrin/agarose hydrogels, PLA scaffolds coated with nanohydroxyapatite or naringin, and alginate–xanthan scaffolds containing copper-doped bioglass nanoparticles.

All included studies used 3D-printing-based fabrication [2,9,11,12,23,33,35,39,43]. Extrusion-based printing predominated [2,9,11,12,23,33,39], typically with nozzle diameters between 0.4 and 0.5 mm. One study used FDM to fabricate PLA scaffolds [43], while another used stereolithography to fabricate endodontic release inserts [35]. Reported pore sizes ranged approximately from 270 to 550 μm, depending on material and printing parameters.

Across most studies, the endodontic application primarily targeted pulp regeneration and/or odontogenic/osteogenic differentiation [2,11,12,23,33,39,43]. In contrast, Leveque et al. [35] focused specifically on early apical release kinetics of molecules from endodontic hydrogels, using 3D-printed inserts, while Ortega et al. [9] explored root canal disinfection using antimicrobial hydrogel scaffolds.

Table 2. Characteristics of the included studies.

Author (Year)	Main Objective	Experimental Model	Bioink/Scaffold Composition	Printing	Endodontic Focus	Conclusion
Cho et al. 2023 [2]	To evaluate whether a leptin-loaded, aminated PCL 3D-printed scaffold enhances adhesion, proliferation, and odontoblastic differentiation of human dental pulp cells.	hDPSCs	A 3D-printed PCL scaffold with 65% porosity and 6 mm disks was produced. Its surface was aminated with 1,6-hexanediamine and then functionalized with leptin using Traut’s reagent and Sulfo-SMCC.	Extrusion-based 3D printing (Dr. in vivo 4D6; 0.4 mm nozzle).	Regenerative endodontics and vital pulp therapy.	Leptin-loaded 3D-printed PCL increased wettability and enhanced dental pulp cell adhesion, proliferation, and odontoblastic differentiation, supporting its potential as a dental pulp transplant material pending further preclinical and clinical validation.
Ortega et al. 2025 [9]	This study describes the development of biocompatible 3D-printed hydrogel scaffolds intended for root canal disinfection.	hDPSCs	A 5% gelatin-based hydrogel was produced with added antimicrobial agents at concentrations between 3.9 and 1000 µg/mL, and corresponding freeze-dried versions were also prepared.	Extrusion-based 3D printing (BIOX6, Cellink®):	Regenerative endodontics.	The scaffolds showed good stability, controlled release, strong antimicrobial activity at 150 and 250 μg/mL, cell viability above 70%, and six-month stability after freeze-drying.
Karkehabadi et al. 2025 [11]	To design, fabricate, and characterize a novel nanocomposite scaffold based on alginate-xanthan containing copper-doped bioactive glass nanoparticles.	Bioactive glass nanoparticles with varying copper concentrations (0, 0.5, 2.5 and 5 wt%).	Alginate-xanthan polymer matrix + mesoporous bioactive glass nanoparticles doped with Cu at 0, 0.5, 2.5, and 5 wt%.	Extrusion-based 3D printing (0.4 mm extrusion width; 12 mm3/s speed; 3-layer scaffolds; 35–45% infill) followed by freeze-drying.	Regenerative endodontics.	Scaffolds containing 2.5 wt% copper-doped bioactive glass performed best, whereas 5 wt% led to nanoparticle aggregation, reduced antioxidant activity, and higher hemolysis, showing that copper is beneficial at low levels but toxic at higher doses.
Yu et al. 2019 [12]	To compare the effects of Alginate/Gelatin (Alg-Gel) hydrogel vs 3D-printed Alg-Gel scaffolds on adhesion, proliferation, and osteogenic/odontogenic differentiation of hDPSCs.	hDPSCs	Hydrogel composed of 20% gelatin + 4% alginate, crosslinked with CaCl2 and glutaraldehyde.	Extrusion-based 3D bioprinting using a Bioplotter (Envisiontec); nozzle 400 µm; 37 °C printing.	Regenerative endodontics evaluation of scaffolds for dentin–pulp complex regeneration.	These results indicate that 3D printed alginate-gelatin scaffolds better support stem cell growth and more effectively promote cell proliferation and differentiation than their non-printed counter parts.
Chiu et al. 2017 [23]	To assess whether an MTA-PCL hybrid 3D scaffold supports osteogenic differentiation of human dental pulp stem cells and how this composite affects the scaffold’s biological performance.	hDPSCs	MTA and PCL hybrid scaffold (60/40 wt%).	Extrusion-based 3D printing (BioScaffolder 3.1; nozzle 500 µm; layer-by-layer deposition).	Regenerative endodontics-pulp tissue regeneration and osteogenic differentiation.	The 3D-printed MTA/PCL scaffold showed excellent physicochemical properties and enhanced osteogenic differentiation, indicating its strong potential as a biomaterial for bone tissue engineering.
Athirasala et al. 2018 [33]	To develop and characterize a dentin-derived extracellular matrix hydrogel bioink for 3D bioprinting of cell-laden scaffolds and evaluate its printability, cytocompatibility, and odontogenic potential.	Mouse odontoblast-like cells and SCAPs	Alginate (3% w/v) blended with insoluble dentin matrix proteins at ratios 2:1, 1:1, and 1:2 (Alg:Dent). Soluble dentin molecules added at 1–100 μg/mL.	Extrusion-based bioprinting using a customized Hyrel3D coaxial nozzle system.	Regenerative endodontics-engineering scaffolds for regeneration of the pulp–dentin complex.	The dentin-derived hybrid bioinks showed good printability, supported cell viability, and enhanced odontogenic differentiation, indicating potential for pulp–dentin and craniofacial tissue regeneration.
Leveque et al. 2024 [35]	This study aims to design a simple and standardized model to identify parameters that influence the early apical release kinetic of molecules from endodontic hydrogels.	3D-printed ERI model	Fibrin hydrogels were produced by combining fibrinogen with sodium chloride, calcium chloride, and a fluorescent tracer, then adding thrombin to yield final fibrin concentrations of 1.5–6 mg/mL.	3D printing (Formlabs 3B printer; 405 nm photopolymer resin; 50 μm layer thickness).	Drug/molecule release in regenerative endodontics.	ERI use enables investigation of the parameters influencing release kinetics from endodontic hydrogels. Further investigations are necessary to evaluate the interaction of these parameters with each other in animal models and clinic.
Ho et al. 2018 [43]	To determine whether a 3D-printed Biodentine-PCL scaffold promotes human dental pulp cell proliferation and odontogenic differentiation, while also assessing its physicochemical characteristics.	hDPSCs	Biodentine + PCL (60 wt% Biodentine in PCL).	Extrusion-based 3D printing (BioScaffolder 3.1; nozzle 500 µm; layer-by-layer deposition).	Regenerative endodontics-odontogenesis/dentin–pulp complex repair.	The composite scaffolds showed Biodentine-like bioactivity and promoted stem cell differentiation, indicating strong potential for dental and bone regeneration.
Dawood & Mahdee, 2025 [39]	To fabricate and characterize 3D-printed PLA porous scaffolds coated with nHA, NAR, or their combination (nHA/NAR), evaluating mechanical properties, antibacterial activity, cytotoxicity, and bioactivity for pulp regeneration.	FDM-printed PLA structures	PLA scaffolds with 300 or 700 µm pores were coated via PVA assisted dip-coating with nHA, NAR, or a combination of both.	Fused deposition modeling 3D printing (Creality CR-10S; 210 °C extrusion; 0.1 mm layer thickness).	Regenerative endodontics/pulp regeneration.	The 3D-printed PLA scaffold coated with nHA and NAR showed improved surface features, mechanical strength, antibacterial effects, and biocompatibility, indicating strong potential for regenerative applications.

CaCl₂: Calcium chloride; ERI: Endodontic Release Inserts; FDM: Fused deposition modeling; hDPSCs: Human dental pulp cells/Human dental pulp stem cells; mL: Milliliter; MTA: Mineral trioxide Aggregate; NAR: Naringin; nHA: NanoHydroxyapatite; PCL: Polycaprolactone; PLA: Poly lactic acid; PVA: Polyvinyl alcohol; SCAP: Stem cells from the apical papilla; Sulfo-SMCC: Sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate; μg: Microgram.

Table 2. Characteristics of the included studies.

Author (Year)	Main Objective	Experimental Model	Bioink/Scaffold Composition	Printing	Endodontic Focus	Conclusion
Cho et al. 2023 [2]	To evaluate whether a leptin-loaded, aminated PCL 3D-printed scaffold enhances adhesion, proliferation, and odontoblastic differentiation of human dental pulp cells.	hDPSCs	A 3D-printed PCL scaffold with 65% porosity and 6 mm disks was produced. Its surface was aminated with 1,6-hexanediamine and then functionalized with leptin using Traut’s reagent and Sulfo-SMCC.	Extrusion-based 3D printing (Dr. in vivo 4D6; 0.4 mm nozzle).	Regenerative endodontics and vital pulp therapy.	Leptin-loaded 3D-printed PCL increased wettability and enhanced dental pulp cell adhesion, proliferation, and odontoblastic differentiation, supporting its potential as a dental pulp transplant material pending further preclinical and clinical validation.
Ortega et al. 2025 [9]	This study describes the development of biocompatible 3D-printed hydrogel scaffolds intended for root canal disinfection.	hDPSCs	A 5% gelatin-based hydrogel was produced with added antimicrobial agents at concentrations between 3.9 and 1000 µg/mL, and corresponding freeze-dried versions were also prepared.	Extrusion-based 3D printing (BIOX6, Cellink®):	Regenerative endodontics.	The scaffolds showed good stability, controlled release, strong antimicrobial activity at 150 and 250 μg/mL, cell viability above 70%, and six-month stability after freeze-drying.
Karkehabadi et al. 2025 [11]	To design, fabricate, and characterize a novel nanocomposite scaffold based on alginate-xanthan containing copper-doped bioactive glass nanoparticles.	Bioactive glass nanoparticles with varying copper concentrations (0, 0.5, 2.5 and 5 wt%).	Alginate-xanthan polymer matrix + mesoporous bioactive glass nanoparticles doped with Cu at 0, 0.5, 2.5, and 5 wt%.	Extrusion-based 3D printing (0.4 mm extrusion width; 12 mm3/s speed; 3-layer scaffolds; 35–45% infill) followed by freeze-drying.	Regenerative endodontics.	Scaffolds containing 2.5 wt% copper-doped bioactive glass performed best, whereas 5 wt% led to nanoparticle aggregation, reduced antioxidant activity, and higher hemolysis, showing that copper is beneficial at low levels but toxic at higher doses.
Yu et al. 2019 [12]	To compare the effects of Alginate/Gelatin (Alg-Gel) hydrogel vs 3D-printed Alg-Gel scaffolds on adhesion, proliferation, and osteogenic/odontogenic differentiation of hDPSCs.	hDPSCs	Hydrogel composed of 20% gelatin + 4% alginate, crosslinked with CaCl2 and glutaraldehyde.	Extrusion-based 3D bioprinting using a Bioplotter (Envisiontec); nozzle 400 µm; 37 °C printing.	Regenerative endodontics evaluation of scaffolds for dentin–pulp complex regeneration.	These results indicate that 3D printed alginate-gelatin scaffolds better support stem cell growth and more effectively promote cell proliferation and differentiation than their non-printed counter parts.
Chiu et al. 2017 [23]	To assess whether an MTA-PCL hybrid 3D scaffold supports osteogenic differentiation of human dental pulp stem cells and how this composite affects the scaffold’s biological performance.	hDPSCs	MTA and PCL hybrid scaffold (60/40 wt%).	Extrusion-based 3D printing (BioScaffolder 3.1; nozzle 500 µm; layer-by-layer deposition).	Regenerative endodontics-pulp tissue regeneration and osteogenic differentiation.	The 3D-printed MTA/PCL scaffold showed excellent physicochemical properties and enhanced osteogenic differentiation, indicating its strong potential as a biomaterial for bone tissue engineering.
Athirasala et al. 2018 [33]	To develop and characterize a dentin-derived extracellular matrix hydrogel bioink for 3D bioprinting of cell-laden scaffolds and evaluate its printability, cytocompatibility, and odontogenic potential.	Mouse odontoblast-like cells and SCAPs	Alginate (3% w/v) blended with insoluble dentin matrix proteins at ratios 2:1, 1:1, and 1:2 (Alg:Dent). Soluble dentin molecules added at 1–100 μg/mL.	Extrusion-based bioprinting using a customized Hyrel3D coaxial nozzle system.	Regenerative endodontics-engineering scaffolds for regeneration of the pulp–dentin complex.	The dentin-derived hybrid bioinks showed good printability, supported cell viability, and enhanced odontogenic differentiation, indicating potential for pulp–dentin and craniofacial tissue regeneration.
Leveque et al. 2024 [35]	This study aims to design a simple and standardized model to identify parameters that influence the early apical release kinetic of molecules from endodontic hydrogels.	3D-printed ERI model	Fibrin hydrogels were produced by combining fibrinogen with sodium chloride, calcium chloride, and a fluorescent tracer, then adding thrombin to yield final fibrin concentrations of 1.5–6 mg/mL.	3D printing (Formlabs 3B printer; 405 nm photopolymer resin; 50 μm layer thickness).	Drug/molecule release in regenerative endodontics.	ERI use enables investigation of the parameters influencing release kinetics from endodontic hydrogels. Further investigations are necessary to evaluate the interaction of these parameters with each other in animal models and clinic.
Ho et al. 2018 [43]	To determine whether a 3D-printed Biodentine-PCL scaffold promotes human dental pulp cell proliferation and odontogenic differentiation, while also assessing its physicochemical characteristics.	hDPSCs	Biodentine + PCL (60 wt% Biodentine in PCL).	Extrusion-based 3D printing (BioScaffolder 3.1; nozzle 500 µm; layer-by-layer deposition).	Regenerative endodontics-odontogenesis/dentin–pulp complex repair.	The composite scaffolds showed Biodentine-like bioactivity and promoted stem cell differentiation, indicating strong potential for dental and bone regeneration.
Dawood & Mahdee, 2025 [39]	To fabricate and characterize 3D-printed PLA porous scaffolds coated with nHA, NAR, or their combination (nHA/NAR), evaluating mechanical properties, antibacterial activity, cytotoxicity, and bioactivity for pulp regeneration.	FDM-printed PLA structures	PLA scaffolds with 300 or 700 µm pores were coated via PVA assisted dip-coating with nHA, NAR, or a combination of both.	Fused deposition modeling 3D printing (Creality CR-10S; 210 °C extrusion; 0.1 mm layer thickness).	Regenerative endodontics/pulp regeneration.	The 3D-printed PLA scaffold coated with nHA and NAR showed improved surface features, mechanical strength, antibacterial effects, and biocompatibility, indicating strong potential for regenerative applications.

CaCl₂: Calcium chloride; ERI: Endodontic Release Inserts; FDM: Fused deposition modeling; hDPSCs: Human dental pulp cells/Human dental pulp stem cells; mL: Milliliter; MTA: Mineral trioxide Aggregate; NAR: Naringin; nHA: NanoHydroxyapatite; PCL: Polycaprolactone; PLA: Poly lactic acid; PVA: Polyvinyl alcohol; SCAP: Stem cells from the apical papilla; Sulfo-SMCC: Sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate; μg: Microgram.

Table 3. Biological, microbiological and physicochemical assays of the included studies.

Author (Year)	Groups	Viability Assays	Differentiation/Markers	Mineralization/Bioactivity	Physicochemical Properties	Antimicrobial Tests
Cho et al. 2023 [2]	PCL Aminated PCL Leptin-loaded PCL	EZ-Cytox (%) 24 h Cell proliferation was significantly higher in the Leptin-loaded PCL group than in both the control and Aminated PCL groups (p < 0.001 and p < 0.0001).	ALP 7 d	Alizarin red S 14 d	The scaffold has 270–340 μm pores, increased hydrophilicity after amination, crystal formation after leptin conjugation, and a controlled leptin release reaching about 96 percent in 14 days.	n.a.
Ortega et al. 2025 [9]	Gel GL5 GL5-Q125 GL5-Q250 GL5-Q500 GL5-Q1000 Freeze-dried FGL5 FGL5-Q125 FGL5-Q250 FGL5-Q500 FGL5-Q1000	MTT assay (%) 24 h, 72 h and 7 d	n.a.	n.a.	The bioinks showed initial elasticity, shear-thinning behavior, and thermal instability near 26 °C. BDMDAC enhanced structural strength and viscosity, forming denser networks. The analysis was descriptive without statistical testing.	CFU/mL MRSA 24 h GL5: ≅107 GL5-Q3.9: ≅107 GL5-Q7.8: ≅107 GL5-Q15: n.a. GL5-Q31: n.a. 24 h FGL5: ≅108 FGL5-Q3.9: ≅108 FGL5-Q7.8: ≅108 FGL5-Q15: n.a. FGL5-Q31: n.a. P. aeruginosa 24 h GL5: ≅107 GL5-Q125 ≅ 107 GL5-Q250: ≅106 *** GL5-Q500: ≅103 *** GL5-Q1000: n.a. 24 h FGL5: ≅109 FGL5-Q125 ≅ 109 FGL5-Q250: ≅108 *** FGL5-Q500: ≅108 *** FGL5-Q1000: n.a. P. gingivalis 24 h GL5: ≅103 GL5-Q15 ≅ 103 GL5-Q31: ≅103 GL5-Q62: n.a. GL5-Q125: n.a. 24 h FGL5: ≅103 FGL5-Q15: ≅103 FGL5-Q31: ≅103 FGL5-Q62: n.a. FGL5-Q125: n.a. E. faecalis 24 h GL5: ≅108 GL5-Q31: ≅197 GL5-Q62: ≅ 106 *** GL5-Q125: n.a. GL5-Q250: n.a. 24 h FGL5: ≅109 FGL5-Q31: ≅107 FGL5-Q62: ≅106 *** FGL5-Q125: n.a. FGL5-Q250: n.a. S. mutans 24 h GL5: ≅107 GL5-Q31: ≅197 GL5-Q62: ≅105 *** GL5-Q125: n.a. GL5-Q250: n.a. 24 h FGL5: ≅107 FGL5-Q31: ≅107 FGL5-Q62: ≅107 FGL5-Q125: n.a. FGL5-Q250: n.a.
Karkehabadi et al. 2025 [11]	B0 B0.5 B2.5 B5	n.a.	n.a.	n.a.	FTIR Copper caused band shifts and reduced OH-band intensity, indicating its incorporation into the glass network. The composite scaffolds showed additional band shifts, suggesting strong polymer bioglass interactions. EDS EDS confirmed copper doping in the nanoparticles (B0.5, B2.5, B5). In the scaffolds, A-X contained C, Ca, O, and N; A-XB0 added Si; and A-XB0.5, A-XB2.5, and A-XB5 incorporated Cu, confirming successful integration. XRD The XRD diffractogram revealed a high degree of similarity among the four nanoparticle samples, indicating that all of them exhibit a predominantly amorphous character.
Yu et al. 2019 [12]	NC Alg-Gel 3 D-printed-Alg-Gel	DSPP The 3D Alg-Gel group showed significantly higher levels than both the NC and Alg-Gel groups, and the conventional Alg-Gel group also exceeded the NC group (p < 0.001). CCK-8 1 d, 3 d and 5 d Cell proliferation was significantly higher in the Alg-Gel and 3D Alg-Gel groups than in NC, with the 3D scaffold showing the best results at both 3 and 5 days (p < 0.001).	Real-time PCR ALP OCN The 3D Alg-Gel group showed significantly higher levels than both the NC and Alg-Gel groups, and the conventional Alg-Gel group also exceeded the NC group (p < 0.001) across both periods and analyses.	Alizarin red S 7 d and 14 d	Elemental analysis Calcium Phosphorus	n.a.
Chiu et al. 2017 [23]	Control PCL MTA/PCL	PrestoBlue 3 h, 6 h, 12 h, 1 d, 3 d and 7 d	ALP 3 d and 7 d OC (pg/mL) 7 d and 14 d	Alizarin red S 0 d, 7 d and 14 d Ion release Si-PCL, Si-MTA/PCL, Ca-PCL and Ca-MTA/PCL 0 h, 1 d, 2 d, 3 d, 4 d, 5 d and 6 d SBF immersion test (bioactivity) Apatite deposition increased from approximately 15% at 0.5 days to about 80% at 7 days.	Pore structure & morphology Pore size ~500 µm; 70% porosity; fully interconnected structure. Mechanical strength (MPa) The scaffold showed a compressive strength of about 4.5 MPa, which increased during the first 30 days in SBF but declined with longer immersion. XRD MTA and PCL retain their original crystalline structure after processing.	n.a.
Athirasala et al. 2018 [33]	Alginate 3% Alg-Dent 2:1, 1:1 and 1:2	Live dead Alginate 3% Alg-Dent 2:1, 1:1 and 1:2 1 d, 3 d and 5 d	ALP 7 d Alg-Dent 1:1 An increase in protein-level ALP activity was observed in the bioinks containing 100 μg/mL compared with the 1:1 Alg-Dent control group (p < 0.05).	n.a.	Pure alginate exhibited a compression modulus of approximately 6 kPa, whereas the addition of 2, 1, or ½ parts of dentin to each part of alginate significantly reduced this value (p < 0.05) to around 1–2 kPa.	n.a.
Leveque et al. 2024 [35]	Apical diameter 0.5 mm, 1.0 mm and 2.0 mm Apical solution PBS and Serum Hydrogel type Agarose 2.5% Fibrin 1.5, 3 and 6 mg/mL Molecule type Fluorescein and BDP-500 Nanoencapsulation: BDP-500 Nanoencapsulated BDP-500	n.a.	n.a.	Apical solution 0 h, 8 h, 16 h and 24 h A statistically significant difference was observed between the solutions (p < 0.001). Hydrogel type 0 h, 8 h, 16 h and 24 h No significant difference was observed between hydrogel types, but reducing fibrin from 3 to 1.5 mg/mL resulted in a significant change (p < 0.001). Molecule type 0 h, 8 h, 16 h and 24 h A statistically significant difference was observed between the solutions (p < 0.05).	Apical diameter 0 h, 8 h, 16 h and 24 h All apical diameters exhibited statistically significant differences between each Other (p < 0.05). Nanoencapsulation 0 h, 8 h, 16 h and 24 h A statistically significant difference was observed between the solutions (p < 0.05).	n.a.
Ho et al. 2018 [43]	Control PCL BD/PCL	PrestoBlue 1 d, 3 d and 7 d	ALP 3 d and 7 d OC (pg/mL) 7 d and 14 d	Alizarin red S 0 d, 7 d and 14 d SBF immersion test (bioactivity) Analyses showed ~500 μm struts and ~550 μm pores, with SBF immersion producing early precipitates and a dense apatite layer by 7 days.	Mechanical strength (MPa) Scaffolds showed ~500 μm struts, 550 μm pores, compressive strength ~6.5 MPa, and rapid apatite formation in SBF.	n.a.
Dawood & Mahdee, 2025 [39]	PLA PLA/nHA PLA/NAR PLA/nHA/NAR	MTT assay The PLA scaffold was biocompatible (p > 0.05), NAR was less cytotoxic than nHA, and the NAR + nHA combination remained biocompatible with 27.15% cytotoxicity and synergistic activity.	n.a.	It was observed that a calcium phosphate apatite layer formed on the surface of the PLA/nHA and PLA/nHA/NAR scaffolds after immersion in SBF.	EDX analysis Pure PLA showed only carbon and oxygen. nHA added calcium and phosphorus, and NAR increased carbon. After SBF immersion, calcium rose in all groups and phosphorus rose only with nHA. No statistical tests were performed. Compressive and flexural strength Compression and Flexural P300 and P700 Strength differed significantly across pore sizes (p < 0.000), but coating types showed no differences when pore size was constant (p < 0.000).	Halo/mm S. mutans E. Faecalis Different superscript letters within each bacterial test indicate statistically significant differences (p < 0.05).

3D: Three-dimensional (3D) printing; Alg-gel: Alginate/gelatin hydrogel; ALP: Alkaline phosphatase; B: Bioactive glass nanoparticles; BD: Biodentine; BDMDAC: Benzyldimethyldodecylammonium chloride; BDP: Hydrophobic; CCK-8: Cell Counting Kit-8; CFU: Colony Forming Unit; d: day; DSPP: Dentin sialophosphoprotein; E. faecalis: Enterococcus faecalis; EDS and EDX: Energy-dispersive X-ray spectroscopy; FGL: Freeze-dried gel; FTIR: Fourier transforms infrared; GL: Gel; kPa: Kilopascal; mg: Milligram; ml: Milliliter; mm: Millimeter; mPA: Megapascal; MRSA: Methicillin-resistant Staphylococcus aureus; MTA: Mineral Trioxide Aggregate; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; n.a.: Not applicable; NAR: Naringin; NC: Negative control; nHa: nano-Hydroxyapatite; OC/OCN: Osteocalcin; OH: Hydroxyl; P. aeruginosa: Pseudomonas aeruginosa; P. gingivalis: Porphyromonas gingivalis; PBS: Phosphate-buffered saline; PCL: Polycaprolactone; PCR: Polymerase chain reaction; PLA: Poly lactic acid; S. mutans: Streptococcus mutans; SBF: Simulated body fluid; XRD: X-ray diffractometry; µm: Micrometer. Significant differences are represented by different letters in each study (p < 0.05). *** Statistically significant difference (p < 0.0001).

Table 3. Biological, microbiological and physicochemical assays of the included studies.

Author (Year)	Groups	Viability Assays	Differentiation/Markers	Mineralization/Bioactivity	Physicochemical Properties	Antimicrobial Tests
Cho et al. 2023 [2]	PCL Aminated PCL Leptin-loaded PCL	EZ-Cytox (%) 24 h Cell proliferation was significantly higher in the Leptin-loaded PCL group than in both the control and Aminated PCL groups (p < 0.001 and p < 0.0001).	ALP 7 d	Alizarin red S 14 d	The scaffold has 270–340 μm pores, increased hydrophilicity after amination, crystal formation after leptin conjugation, and a controlled leptin release reaching about 96 percent in 14 days.	n.a.
Ortega et al. 2025 [9]	Gel GL5 GL5-Q125 GL5-Q250 GL5-Q500 GL5-Q1000 Freeze-dried FGL5 FGL5-Q125 FGL5-Q250 FGL5-Q500 FGL5-Q1000	MTT assay (%) 24 h, 72 h and 7 d	n.a.	n.a.	The bioinks showed initial elasticity, shear-thinning behavior, and thermal instability near 26 °C. BDMDAC enhanced structural strength and viscosity, forming denser networks. The analysis was descriptive without statistical testing.	CFU/mL MRSA 24 h GL5: ≅107 GL5-Q3.9: ≅107 GL5-Q7.8: ≅107 GL5-Q15: n.a. GL5-Q31: n.a. 24 h FGL5: ≅108 FGL5-Q3.9: ≅108 FGL5-Q7.8: ≅108 FGL5-Q15: n.a. FGL5-Q31: n.a. P. aeruginosa 24 h GL5: ≅107 GL5-Q125 ≅ 107 GL5-Q250: ≅106 *** GL5-Q500: ≅103 *** GL5-Q1000: n.a. 24 h FGL5: ≅109 FGL5-Q125 ≅ 109 FGL5-Q250: ≅108 *** FGL5-Q500: ≅108 *** FGL5-Q1000: n.a. P. gingivalis 24 h GL5: ≅103 GL5-Q15 ≅ 103 GL5-Q31: ≅103 GL5-Q62: n.a. GL5-Q125: n.a. 24 h FGL5: ≅103 FGL5-Q15: ≅103 FGL5-Q31: ≅103 FGL5-Q62: n.a. FGL5-Q125: n.a. E. faecalis 24 h GL5: ≅108 GL5-Q31: ≅197 GL5-Q62: ≅ 106 *** GL5-Q125: n.a. GL5-Q250: n.a. 24 h FGL5: ≅109 FGL5-Q31: ≅107 FGL5-Q62: ≅106 *** FGL5-Q125: n.a. FGL5-Q250: n.a. S. mutans 24 h GL5: ≅107 GL5-Q31: ≅197 GL5-Q62: ≅105 *** GL5-Q125: n.a. GL5-Q250: n.a. 24 h FGL5: ≅107 FGL5-Q31: ≅107 FGL5-Q62: ≅107 FGL5-Q125: n.a. FGL5-Q250: n.a.
Karkehabadi et al. 2025 [11]	B0 B0.5 B2.5 B5	n.a.	n.a.	n.a.	FTIR Copper caused band shifts and reduced OH-band intensity, indicating its incorporation into the glass network. The composite scaffolds showed additional band shifts, suggesting strong polymer bioglass interactions. EDS EDS confirmed copper doping in the nanoparticles (B0.5, B2.5, B5). In the scaffolds, A-X contained C, Ca, O, and N; A-XB0 added Si; and A-XB0.5, A-XB2.5, and A-XB5 incorporated Cu, confirming successful integration. XRD The XRD diffractogram revealed a high degree of similarity among the four nanoparticle samples, indicating that all of them exhibit a predominantly amorphous character.
Yu et al. 2019 [12]	NC Alg-Gel 3 D-printed-Alg-Gel	DSPP The 3D Alg-Gel group showed significantly higher levels than both the NC and Alg-Gel groups, and the conventional Alg-Gel group also exceeded the NC group (p < 0.001). CCK-8 1 d, 3 d and 5 d Cell proliferation was significantly higher in the Alg-Gel and 3D Alg-Gel groups than in NC, with the 3D scaffold showing the best results at both 3 and 5 days (p < 0.001).	Real-time PCR ALP OCN The 3D Alg-Gel group showed significantly higher levels than both the NC and Alg-Gel groups, and the conventional Alg-Gel group also exceeded the NC group (p < 0.001) across both periods and analyses.	Alizarin red S 7 d and 14 d	Elemental analysis Calcium Phosphorus	n.a.
Chiu et al. 2017 [23]	Control PCL MTA/PCL	PrestoBlue 3 h, 6 h, 12 h, 1 d, 3 d and 7 d	ALP 3 d and 7 d OC (pg/mL) 7 d and 14 d	Alizarin red S 0 d, 7 d and 14 d Ion release Si-PCL, Si-MTA/PCL, Ca-PCL and Ca-MTA/PCL 0 h, 1 d, 2 d, 3 d, 4 d, 5 d and 6 d SBF immersion test (bioactivity) Apatite deposition increased from approximately 15% at 0.5 days to about 80% at 7 days.	Pore structure & morphology Pore size ~500 µm; 70% porosity; fully interconnected structure. Mechanical strength (MPa) The scaffold showed a compressive strength of about 4.5 MPa, which increased during the first 30 days in SBF but declined with longer immersion. XRD MTA and PCL retain their original crystalline structure after processing.	n.a.
Athirasala et al. 2018 [33]	Alginate 3% Alg-Dent 2:1, 1:1 and 1:2	Live dead Alginate 3% Alg-Dent 2:1, 1:1 and 1:2 1 d, 3 d and 5 d	ALP 7 d Alg-Dent 1:1 An increase in protein-level ALP activity was observed in the bioinks containing 100 μg/mL compared with the 1:1 Alg-Dent control group (p < 0.05).	n.a.	Pure alginate exhibited a compression modulus of approximately 6 kPa, whereas the addition of 2, 1, or ½ parts of dentin to each part of alginate significantly reduced this value (p < 0.05) to around 1–2 kPa.	n.a.
Leveque et al. 2024 [35]	Apical diameter 0.5 mm, 1.0 mm and 2.0 mm Apical solution PBS and Serum Hydrogel type Agarose 2.5% Fibrin 1.5, 3 and 6 mg/mL Molecule type Fluorescein and BDP-500 Nanoencapsulation: BDP-500 Nanoencapsulated BDP-500	n.a.	n.a.	Apical solution 0 h, 8 h, 16 h and 24 h A statistically significant difference was observed between the solutions (p < 0.001). Hydrogel type 0 h, 8 h, 16 h and 24 h No significant difference was observed between hydrogel types, but reducing fibrin from 3 to 1.5 mg/mL resulted in a significant change (p < 0.001). Molecule type 0 h, 8 h, 16 h and 24 h A statistically significant difference was observed between the solutions (p < 0.05).	Apical diameter 0 h, 8 h, 16 h and 24 h All apical diameters exhibited statistically significant differences between each Other (p < 0.05). Nanoencapsulation 0 h, 8 h, 16 h and 24 h A statistically significant difference was observed between the solutions (p < 0.05).	n.a.
Ho et al. 2018 [43]	Control PCL BD/PCL	PrestoBlue 1 d, 3 d and 7 d	ALP 3 d and 7 d OC (pg/mL) 7 d and 14 d	Alizarin red S 0 d, 7 d and 14 d SBF immersion test (bioactivity) Analyses showed ~500 μm struts and ~550 μm pores, with SBF immersion producing early precipitates and a dense apatite layer by 7 days.	Mechanical strength (MPa) Scaffolds showed ~500 μm struts, 550 μm pores, compressive strength ~6.5 MPa, and rapid apatite formation in SBF.	n.a.
Dawood & Mahdee, 2025 [39]	PLA PLA/nHA PLA/NAR PLA/nHA/NAR	MTT assay The PLA scaffold was biocompatible (p > 0.05), NAR was less cytotoxic than nHA, and the NAR + nHA combination remained biocompatible with 27.15% cytotoxicity and synergistic activity.	n.a.	It was observed that a calcium phosphate apatite layer formed on the surface of the PLA/nHA and PLA/nHA/NAR scaffolds after immersion in SBF.	EDX analysis Pure PLA showed only carbon and oxygen. nHA added calcium and phosphorus, and NAR increased carbon. After SBF immersion, calcium rose in all groups and phosphorus rose only with nHA. No statistical tests were performed. Compressive and flexural strength Compression and Flexural P300 and P700 Strength differed significantly across pore sizes (p < 0.000), but coating types showed no differences when pore size was constant (p < 0.000).	Halo/mm S. mutans E. Faecalis Different superscript letters within each bacterial test indicate statistically significant differences (p < 0.05).

3D: Three-dimensional (3D) printing; Alg-gel: Alginate/gelatin hydrogel; ALP: Alkaline phosphatase; B: Bioactive glass nanoparticles; BD: Biodentine; BDMDAC: Benzyldimethyldodecylammonium chloride; BDP: Hydrophobic; CCK-8: Cell Counting Kit-8; CFU: Colony Forming Unit; d: day; DSPP: Dentin sialophosphoprotein; E. faecalis: Enterococcus faecalis; EDS and EDX: Energy-dispersive X-ray spectroscopy; FGL: Freeze-dried gel; FTIR: Fourier transforms infrared; GL: Gel; kPa: Kilopascal; mg: Milligram; ml: Milliliter; mm: Millimeter; mPA: Megapascal; MRSA: Methicillin-resistant Staphylococcus aureus; MTA: Mineral Trioxide Aggregate; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide; n.a.: Not applicable; NAR: Naringin; NC: Negative control; nHa: nano-Hydroxyapatite; OC/OCN: Osteocalcin; OH: Hydroxyl; P. aeruginosa: Pseudomonas aeruginosa; P. gingivalis: Porphyromonas gingivalis; PBS: Phosphate-buffered saline; PCL: Polycaprolactone; PCR: Polymerase chain reaction; PLA: Poly lactic acid; S. mutans: Streptococcus mutans; SBF: Simulated body fluid; XRD: X-ray diffractometry; µm: Micrometer. Significant differences are represented by different letters in each study (p < 0.05). *** Statistically significant difference (p < 0.0001).

3.3. Viability Assays

Seven studies [2,9,12,23,33,39,43] evaluated viability and/or metabolic activity using assays such as PrestoBlue, Cell Counting Kit-8 (CCK-8), EZ-Cytox, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and Live/Dead staining. Overall, viability tended to increase over time (from early time points up to 7 days), particularly in bioactive or functionalized formulations (e.g., containing MTA, Biodentine, leptin, naringin, and/or nHA) and in 3D-printed constructs.

PrestoBlue-based analyses [23,42] showed progressive increases in metabolic activity from ~3 h to 7 days, with MTA/PCL and BD/PCL demonstrating significantly higher viability than pure PCL and control groups. Live/Dead staining [33] and CCK-8 assays [12] confirmed high early viability in hydrogel systems and maintenance above 70% up to 5 days; notably, 3D-printed alginate-gelatin scaffolds exhibited marked increases at days 3 and 5.

Using EZ-Cytox, ref. [2] reported a pronounced increase in viability at 24 h in leptin-loaded PCL (approximately 140% relative to reference conditions), exceeding the performance of both PCL and aminated PCL. MTT assays [9,43] (indicated overall biocompatibility) of PLA-based scaffolds and naringin-containing formulations, as well as a synergistic effect when naringin was combined with nHA. In Ortega et al. [9], increasing concentrations of benzyldimethyldodecylammonium chloride (BDMDAC) reduced viability in a dose-dependent manner: concentrations up to 250 μg/mL maintained high viability up to 7 days, whereas concentrations ≥ 500 μg/mL were associated with pronounced cytotoxicity.

3.4. Differentiation and Marker Expression

Five studies [2,12,23,33,43] evaluated odontogenic and/or osteogenic differentiation using multiple markers, collectively showing increased differentiation-related activity between 3 and 14 days, irrespective of the specific assay employed.

Alkaline phosphatase (ALP) activity increased progressively between days 3 and 7, with bioactive composites such as MTA/PCL and BD/PCL [23,43] presenting significantly higher ALP levels than controls and pure PCL. These studies also reported increased osteocalcin expression, particularly at 14 days.

Dentin-containing formulations [33] and 3D-printed alginate–gelatin scaffolds [12] further enhanced differentiation. Specifically, Yu et al. [12] reported very high expression of dentin sialophosphoprotein (DSPP), ALP, and osteocalcin (OCN) in printed scaffolds compared with conventional groups. Similarly, leptin-loaded PCL scaffolds [2] demonstrated increased ALP activity at 7 days. More recent studies focusing on NAR, nHA, or BDMDAC [9,43] did not assess differentiation markers.

3.5. Mineralization and Bioactivity

Five studies [2,12,23,39,43] assessed mineralization and/or bioactivity using different methodological approaches but reported convergent trends. Overall, biomaterials showed progressive mineral deposition and enhanced apatite-forming ability, particularly when incorporating bioactive components (MTA, BD, NAR, nHA) and/or when fabricated as 3D-printed constructs, typically between 7 and 14 days.

Alizarin Red staining indicated that MTA/PCL and BD/PCL [23,42] generated significantly greater mineral nodule formation than pure PCL at both evaluated time points. Yu et al. [12] reported the highest mineral deposition in 3D-printed alginate-gelatin scaffolds, accompanied by increased calcium and phosphorus content. Modified PCL formulations (aminated PCL and leptin-loaded PCL) exhibited modest mineralization, yet consistently higher than pure PCL at 14 days [2]. Bioactivity assays based on immersion in simulated body fluid (SBF) supported these findings, showing progressive and, in some cases, rapid apatite formation across compositions.

Although Leveque et al. [35] was listed under mineralization/bioactivity in summary tables, that study did not directly evaluate mineral deposition; instead, it assessed molecular diffusion through hydrogels and periapical solutions. Its inclusion reflects the relevance of diffusion kinetics for bioactivity-related processes, even though it does not constitute a direct mineralization analysis.

3.6. Physicochemical Properties

All studies [2,9,11,12,23,33,35,39,43] reported physicochemical outcomes indicating overall stability, appropriate porous architecture, and, where evaluated, the ability to form apatite in SBF, particularly in formulations containing bioactive phases or structural modifications.

In MTA/PCL and BD/PCL scaffolds [23,43], pores ranged from ~500 to 550 µm, compressive strength ranged from ~4.5 to 6.5 MPa, and the crystalline structure was preserved after processing. These scaffolds showed evident apatite formation after SBF immersion, including dense surface layers by day 7. In alginate-based systems [33], incorporation of dentin reduced the compressive modulus from approximately 6 kPa to ~1–2 kPa. In modified PCL scaffolds [2], pores were reported between 270 and 340 µm; amination increased hydrophilicity, leptin incorporation influenced crystal formation, and controlled release of the biomolecule was observed over 14 days.

PLA formulations coated with nHA and/or NAR [43] showed compositional signatures consistent with the applied coatings, increased calcium content, and, when hydroxyapatite was present, increased phosphorus after SBF immersion. Mechanical properties varied significantly across pore sizes, while coating effects were less pronounced when pore size was controlled. Copper-doped scaffolds [11] exhibited spectroscopic changes consistent with ionic incorporation; integration was supported by energy-dispersive X-ray spectroscopy (EDS), and amorphous diffraction patterns were observed.

3.7. Antimicrobial Tests

Only two studies [9,39] evaluated antimicrobial performance. Overall, greater bacterial reduction was observed in functionalized biomaterials containing bioactive or antimicrobial components, whereas base formulations exhibited limited activity.

Dawood and Mahdee [39] reported that, against Streptococcus mutans and Enterococcus faecalis, PLA/nHA formulations, and particularly PLA/nHA/NAR, produced significantly greater bacterial reduction compared with pure PLA and PLA/NAR. The effect was more pronounced against Enterococcus faecalis (p < 0.05), suggesting a central contribution of nHA to antimicrobial activity, further enhanced by association with naringin.

Ortega et al. [9] demonstrated a dose-dependent antimicrobial response for BDMDAC-containing hydrogels. Low concentrations produced minimal antibacterial effects, whereas intermediate to high concentrations (Q250–Q500) markedly reduced bacterial load, especially for Pseudomonas aeruginosa (p < 0.0001). Very high concentrations (≥Q1000) could not be fully assessed due to cytotoxicity, highlighting practical safety constraints for this compound.

3.8. Intra-Study Quantitative Assessment

An intra-study quantitative analysis was performed using data from Dawood and Mahdee [39]. Relative to PLA, all modified scaffolds showed statistically significant increases in inhibition halo diameters against S. mutans and E. faecalis. For S. mutans, PLA/NAR increased the halo by 4.2 mm (95% CI: 1.9–6.5), PLA/nHA by 2.4 mm (95% CI: 1.5–3.3), and PLA/nHA/NAR by 8.0 mm (95% CI: 3.2–12.8). For E. faecalis, increases were 10.6 mm (95% CI: 6.4–14.8) for PLA/NAR, 4.2 mm (95% CI: 2.4–6.0) for PLA/nHA, and 13.0 mm (95% CI: 8.4–17.6) for PLA/nHA/NAR. Collectively, these results indicate that naringin, alone or combined with nHA, substantially enhances antimicrobial performance compared with pure PLA (Figure 2).

3.9. Risk of Bias Assessment of the Included Studies

Risk of bias was assessed using the QUIN tool, which encompasses twelve methodological domains and is summarized in Figure 3 and Tables S3 and S4. Overall, the included studies exhibited a moderate risk of bias, with a total score of 14/24 (58.33%).

In general, studies consistently met domains related to clearly defined objectives, detailed methodological descriptions, appropriate outcome measurement methods, statistical analyses, and reporting of results, which received maximal scores. In contrast, domains associated with bias control were systematically underreported. None of the studies described randomization procedures, blinding of outcome assessors, or operator-related information, and sampling methods were typically insufficiently detailed. Similarly, formal sample size calculations were largely absent, with the exception of Leveque et al. [35], which justified the number of replicates. Notably, in that study, the description of the comparison group was less detailed, resulting in a score of zero for that domain and an overall score consistent with the remaining studies.

Taken together, these findings indicate that although methodologies and outcomes were generally well described, the studies share structural limitations in planning and reporting core elements required to minimize bias. This supports the consistent classification of a moderate risk of bias across the included literature.

4. Discussion

This systematic review indicates substantial progress in the application of 3D-printed/bioprinted scaffolds and bioinks in endodontics, with increasing diversification of materials, biological models, and experimental strategies [2,9,11,12,23,33,35,39,43]. Across studies, these biomaterials showed potential to support cell viability, promote odontogenic differentiation, induce mineralization, and maintain suitable physicochemical properties, while selected formulations also demonstrated antimicrobial effects [2,9,12,23,33,39].

Taken together, although the included studies demonstrate the potential of 3D-printed/bioprinted scaffolds to support cell viability, differentiation, and mineralization, these outcomes should be interpreted considering the substantial heterogeneity in biomaterial composition, scaffold design, cell sources, and experimental conditions [22,23,27]. This variability limits direct comparability and may partially explain the differences observed across studies.

The high inter-reviewer agreement during screening, reflected by near-perfect kappa values, strengthens the methodological reliability of the selection process [37,38]. This is particularly relevant in a research area characterized by methodological heterogeneity that may compromise reproducibility.

Despite variability in biomaterials and experimental goals, the predominance of hDPSCs aligns with their widespread use as a reference model for pulp regeneration investigations [2,9,12,23,39]. The inclusion of SCAPs [33] broadens the biological scope, while studies centered on diffusion and release kinetics [35,43] contribute mechanistic insights relevant to bioactivity and therapeutic delivery. However, differences in cellular models, particularly between hDPSCs and SCAPs, may influence the biological outcomes, as these populations present distinct proliferative capacities and differentiation potentials, which should be considered when comparing results across studies.

Regarding viability, a consistent time-dependent increase was observed, particularly for compositions incorporating MTA, BD, leptin, NAR, and/or nHA and for printed constructs. Three-dimensional architectures are known to support adhesion, proliferation, and differentiation by providing biomimetic microenvironments [44,45]. These findings are consistent with the controlled architecture enabled by 3D printing, as pore sizes around ~500 μm have been widely associated with improved cell infiltration, nutrient diffusion, and tissue formation, while supporting osteogenesis and angiogenesis [22,46,47]. The relative consistency observed in viability outcomes across studies is likely associated with the presence of bioactive and ion-releasing materials, as well as the structural advantages provided by 3D architectures. Nevertheless, variations in scaffold composition, printing resolution, and culture conditions may account for differences in the magnitude of these effects.

Differentiation outcomes were likewise consistent, with increases in markers such as ALP and OCN typically between 7 and 14 days, especially in scaffolds containing calcium silicate-based components or produced via 3D printing [2,12,23,33,39]. Calcium silicate-based materials (e.g., MTA and Biodentine) have documented bioactive properties that can modulate odontogenic/osteogenic pathways and enhance ALP activity, OCN expression, and mineral formation [48,49,50]. These effects are often linked to ion release (calcium and silicon) and apatite formation, supporting the observed performance of MTA/PCL and BD/PCL composites [22,51].

These effects can be explained by the release of calcium and silicon ions, which are known to activate signaling pathways involved in odontogenic and osteogenic differentiation. Such ionic interactions regulate gene expression related to mineralization, including markers such as ALP and OCN, thereby contributing to the consistent differentiation patterns observed across studies.

Mineralization and bioactivity analyses indicated robust apatite-forming capacity, particularly in compositions containing MTA, BD, NAR, and/or nHA. BD/PCL scaffolds exhibited rapid apatite formation in SBF and progressive mechanical changes during immersion, consistent with the behavior of calcium silicate-based bioceramics [22,52,53]. These properties complement MTA’s reported capacity to harden and increase mechanical strength during the initial days after placement [54]. The combination of bioceramics with polymers may also influence degradation kinetics, as reported for PCL-based composites [55,56], which warrants investigation in longer-term studies.

These findings suggest that bioactivity is governed by a combination of physicochemical and structural factors, in which ion release drives apatite formation, while scaffold architecture modulates fluid exchange and surface interactions. This dual mechanism may explain the robust mineralization observed in calcium silicate-containing scaffolds.

Physicochemical data indicated that 3D-printed scaffolds can maintain stable and suitable architectures for tissue development. MTA- or BD-containing scaffolds displayed pores around 500–550 μm and compressive strength compatible with biological applications. In contrast, hydrogels incorporating dentin exhibited lower compressive modulus, approaching the mechanical range of pulp-like tissues [33]. Chemical modifications (e.g., PCL amination, leptin loading) improved hydrophilicity and enabled controlled release [2]. Additionally, nHA coatings and copper doping were associated with ionic integration and compositional changes after SBF immersion, supporting bioactivity [11].

These physicochemical findings help explain why composite systems, particularly those combining calcium silicate-based bioactive phases with printable polymeric matrices, have emerged as the most promising approaches, as they promote a balance between structural properties, bioactivity, and biological performance [2,11,12,23,33,39]. In contrast, isolated material modifications or acellular systems, although useful for elucidating specific mechanisms, do not yet demonstrate the same level of integrated regenerative performance [11,35,43]. Thus, the most promising strategy involves the development of multifunctional composite scaffolds that integrate structural support, bioactive ion release, and, when necessary, targeted biological or antimicrobial functionalization.

Therefore, the biological performance of these scaffolds should be interpreted as the result of a synergistic interaction between structural design and material composition, where pore architecture facilitates cell infiltration and nutrient diffusion, while chemical modifications regulate surface properties and ion exchange.

From a translational perspective, these findings allow us to outline the key characteristics of an ideal scaffold for regenerative endodontics. Based on the available evidence, the ideal scaffold for regenerative endodontics should combine an interconnected porous architecture with high biocompatibility, supporting cell infiltration, viability, and differentiation [22,44,45,46,47]. It should incorporate bioactive components capable of releasing therapeutic ions to promote mineralization [48,49,50,51], while maintaining adequate physicochemical stability and controlled degradation [52,53,54,55,56]. Importantly, the scaffold should also allow functionalization to introduce antimicrobial or bioactive agents [9,43,57,58,59,60]. Overall, current evidence supports multifunctional composite scaffolds that integrate structural support, bioactivity, and therapeutic functionality [2,11,22,33,39].

Antimicrobial outcomes were evaluated in only two studies, and activity was more pronounced in functionalized biomaterials (nHA/NAR or BDMDAC), whereas base formulations showed limited effects [9,43]. This limited evidence highlights a critical gap in current research, particularly considering that persistent infection remains one of the main causes of failure in regenerative endodontic procedures.

The variability in antimicrobial performance across studies may be attributed to differences in scaffold composition, degree of functionalization, and testing methodologies, including the microbial strains used and experimental conditions [57,58,59]. Notably, bioactive and ion-releasing materials alone may not be sufficient to achieve predictable antimicrobial effects, reinforcing the need for targeted functionalization strategies [60].

Furthermore, the lack of standardized microbiological models limits the comparability of results and hinders the establishment of clear conclusions regarding antimicrobial efficacy. From a clinical perspective, scaffold development should not focus solely on regenerative potential but also incorporate antimicrobial properties to help control residual infection within the root canal system [60,61]. Equally important, however, is prior canal disinfection through appropriate intracanal medication, which remains a critical step for reducing the microbial load before scaffold placement. This combined approach should be emphasized when translating these strategies into clinical practice.

Risk-of-bias evaluation indicated a moderate risk across all studies, largely due to missing information on randomization, blinding, sample-size calculation, and operator details. Although methodologies and results were generally well reported, these limitations reduce the strength of the evidence and underscore the importance of improved standardization in vitro research. These methodological limitations not only affect internal validity but also contribute to the heterogeneity of findings, further complicating the interpretation and comparability of results across studies.

Beyond the intrinsic limitations of the included studies, the present review was constrained by substantial heterogeneity in biomaterials, cell models, printing methods, and experimental time points. Although an intra-study quantitative analysis was feasible for [43], this does not constitute a meta-analysis. A meta-analysis requires at least two studies with sufficiently comparable outcomes, units, and variance estimates, which were not available. The lack of variability statistics in most articles further precluded pooled quantitative synthesis. Accordingly, the quantitative results reported here should be interpreted as complementary to the narrative synthesis rather than as aggregated evidence. Therefore, the absence of standardized outcome measures and comparable datasets reinforces the need for caution when interpreting the available evidence and underscores the current limitations in generating high-level quantitative synthesis.

Despite the promising results, several challenges remain for the clinical implementation of 3D-printed and bioprinted scaffolds in endodontic practice. These include the complexity of scaffold fabrication, high production costs, limited standardization of protocols, and regulatory constraints [2,11,33,35,39]. In addition, the translation of these technologies to the clinical setting requires simplified and reproducible procedures that can be integrated into routine endodontic workflows [9,23,43].

Nevertheless, these advances have the potential to significantly change the current paradigm of regenerative endodontics, shifting from conventional disinfection-based approaches toward biologically driven strategies focused on tissue regeneration [2,12,23,33,39]. If successfully translated into clinical practice, bioprinted scaffolds may enable more predictable and personalized regenerative therapies.

Future research should emphasize methodological standardization, incorporate advanced biological models (e.g., co-cultures, microfluidic systems, organoids), and include long-term degradation and in vivo validation to better assess regenerative efficacy under clinically relevant conditions. Strengthening these aspects will expand the evidence base and facilitate translation of bioprinted endodontic scaffolds into clinical practice. Addressing these challenges will be essential to reduce heterogeneity, improve reproducibility, and enable a more robust translation of experimental findings into clinically applicable regenerative endodontic strategies.

In addition, emerging technologies such as artificial intelligence (AI) and machine learning may further enhance the development of bioprinted scaffolds in endodontics. These approaches have the potential to optimize scaffold design, improve printing parameters, and predict biological responses, thereby increasing precision and reproducibility [62].

In dentistry, AI-assisted bioprinting has already been proposed as a promising strategy to enable personalized regenerative therapies and improve the integration of engineered tissues [63]. Furthermore, machine learning techniques may support the optimization of material selection and structural properties, reducing trial-and-error approaches and accelerating scaffold development [64]. Although still in early stages, the integration of AI with bioprinting technologies may significantly impact the future of regenerative endodontic procedures.

5. Conclusions

Overall, the included studies suggest that 3D-printed/bioprinted scaffolds and bioinks can enhance cell viability, support differentiation, promote mineralization, and maintain adequate bioactivity and physicochemical stability. Formulations incorporating MTA, BD, nHA, and/or NAR generally demonstrated the most favorable performance. Antimicrobial properties were evaluated in only a minority of studies but were more evident in functionalized materials. The intra-study quantitative analysis further supported greater bacterial inhibition in PLA/NAR and PLA/nHA/naringin formulations compared with pure PLA.