OBJECTIVE: To synthesize casein enzymatic hydrolysate (CEH)-laden gelatin methacryloyl (GelMA) fibrous scaffolds and evaluate the cytocompatibility and anti-inflammatory effects on dental pulp stem cells (DPSCs).
METHODS: GelMA fibrous scaffolds with 10%, 20%, and 30% CEH (w/w) and without CEH (control) were obtained via electrospinning. Chemo-morphological, degradation, and mechanical analyses were conducted to evaluate the morphology and composition of the fibers, mass loss, and mechanical properties, respectively. Adhesion/spreading and viability of DPSCs seeded on the scaffolds were also assessed. The anti-inflammatory potential on DPSCs was tested after the chronic challenge of cells with lipopolysaccharides (LPS), followed by treatment with extracts obtained after immersing the scaffolds in α-MEM. The synthesis of the pro-inflammatory cytokines IL-6, IL-1α, and TNF-α was measured by ELISA. Data were analyzed by ANOVA/post-hoc tests (α = 5%).
RESULTS: CEH-laden electrospun fibers had a larger diameter than pure GelMA (p ≤ 0.036). GelMA scaffolds laden with 20% and 30% CEH had a greater mass loss. Tensile strength was reduced for the 10% CEH fibers (p = 0.0052), whereas no difference was observed for the 20% and 30% fibers (p ≥ 0.6736) compared to the control. Young\'s modulus decreased with CEH (p < 0.0001). Elongation at break increased for the 20% and 30% CEH scaffolds (p ≤ 0.0038). Over time, DPSCs viability increased across all groups, indicating cytocompatibility, with CEH-laden scaffolds exhibiting greater cell viability after seven days (p ≤ 0.0166). Also, 10% CEH-GelMA scaffolds decreased the IL-6, IL-1α, and TNF-α synthesis (p ≤ 0.035).
CONCLUSIONS: CEH-laden GelMA scaffolds facilitated both adhesion and proliferation of DPSCs, and 10% CEH provided anti-inflammatory potential after chronic LPS challenge.
CONCLUSIONS: CEH incorporated in GelMA fibrous scaffolds demonstrated the potential to be used as a cytocompatible and anti-inflammatory biomaterial for vital pulp therapy.

This study aimed to perform clinical and radiographic investigations of the effect of regenerative endodontic procedures (REPs) with and without concentrated growth factor (CGF). Fifty-six non-vital and immature teeth from 56 patients were randomly categorized into two groups. Following chemical and mechanical preparation, REPs with and without CGF as a scaffold was induced in the blood clot (BLC) group and the CGF group. All patients were clinically and radiographically evaluated at 6-month and 12-month intervals to monitor their progress and treatment outcomes. When considering the total number of patients, the follow-up rate was 96.4% (54 out of 56 patients) over a 12-month period. Favorable clinical and radiographic outcomes were observed in 92.6% of patients (25 out of 27) in both the CGF and BLC groups; there were no significant differences between the two groups in these respects (p > 0.05). Notable differences were, however, observed in radiographic measurements relating to the development of root length and radiographic root area when compared between the CGF and BLC groups at both the 6-month and 12-month follow-up intervals (p < 0.05). REPs have been proven to represent a conservative and effective approach for promoting maturogenesis in non-vital and immature teeth. Furthermore, the incorporation of CGF as scaffolds holds promising potential for enhancing the desired biological outcomes of this regenerative technique. These findings highlight the clinical significance and potential benefits of CGF supplementation in REPs, further supporting its application in the field of endodontics.

BACKGROUND: Regenerative endodontics involves the use of various root canal medicaments and scaffolds, which may cause crown discoloration.
OBJECTIVE: This study aimed to investigate the combined crown discoloration of scaffolds [platelet-rich fibrin (PRF) and blood clot] applied after administration of different medicaments [modified triple antibiotic paste including doxycycline (mTAPd), modified double antibiotic paste (mDAP), calcium hydroxide (CH), and propolis].
METHODS: In total, 100 human mandibular premolar teeth were selected and prepared. The teeth were apically resected to simulate immature teeth. The positive and negative control groups (n = 10) consisted solely of blood-only and serum-only samples. The remaining 80 teeth were used for the experimental groups with four different medicaments. Three weeks later, either blood or PRF was applied as a scaffold after removing the medicaments (n = 10). Color changes were assessed before medication placement and at the end of the first, second, and third weeks, as well as on days 0, 1, 30, 60, and 90 after scaffold application. Analysis was carried out using repeated measures of variance, Friedman, one-way ANOVA, Kruskal-Wallis, the dependent paired t-test, and Wilcoxon test.
RESULTS: Statistical significance was determined at P = 0.05. All groups including blood and the group including propolis and PRF combination, resulted in a significant increase in discoloration (P < 0.05) and discoloration exceeding clinically acceptable thresholds.
CONCLUSIONS: CH and the modified versions of TAP (mTAPd) and DAP (mDAP) demonstrated an acceptable level of discoloration when used with a combination of PRF at day 90.

Regenerative endodontics aims to restore pulp tissues, thus preserving the vitality of the tooth. One promising approach involves the utilization of decellularized human dental pulp (DHDP) as a scaffold repopulated with Wharton\'s Jelly mesenchymal stem cells (WJMSCs). This study aimed to regenerate pulp tissues using DHDP and WJMSCs following pulpectomy in mature canine teeth of a feline animal model and to investigate the histological features of the regenerated pulp. A 12-month-old male domestic shorthaired felines were used as subjects. Teeth were categorized into untreated (Group 1), pulpectomy with mineral trioxide aggregate (MTA) (Group 2), and pulpectomy with DHDP-repopulated scaffold and MTA (Group 3). The animals were sacrificed six weeks post-intervention. H&E and immunohistochemistry using anti-collagen type 1 and laminin antibodies were used to stain the tissue sections. Histological examinations presented pulp-like tissues in Group 3, with tissue components similar to the structures found in Group 1. Immunohistochemical analysis demonstrated the presence of collagen type I and laminin within the regenerated tissues. The root canals of teeth in Group 2 were devoid of pulpal tissue. DHDP with WJMSCs can potentially be used for pulp regeneration, supporting the modality for developing new clinical protocols in stem cell therapy.

Regenerative endodontics (REP) is a new clinical modality aiming to regenerate damaged soft and hard dental tissues, allowing for root completion in young adults\' teeth. Effective disinfection is crucial for REP success, but commonly used antimicrobials often harm the niche dental pulp stem cells (DPSCs). To our knowledge, this is the first study to explore the biocompatibility and antimicrobial potential of pectin as a potential natural intracanal medicament for REPs. Low methoxyl commercial citrus pectin (LM) (pectin CU701, Herbstreith&Fox.de) was used in all experiments. The pectin\'s antibacterial activity against single species biofilms (E. faecalis and F. nucleatum) was assessed using growth curves. The pectin\'s antimicrobial effect against mature dual-species biofilm was also evaluated using confocal laser scanning microscopy (CLSM) after 30 min and 7 days of treatment. The DPSC biocompatibility with 2% and 4% w/v of the pectin coatings was evaluated using live/dead staining, LDH, and WST-1 assays. Pectin showed a concentration-dependent inhibitory effect against single-species biofilms (E. faecalis and F. nucleatum) but failed to disrupt dual-species biofilm. Pectin at 2% w/v concentration proved to be biocompatible with the HDPSCs. However, 4% w/v pectin reduced both the viability and proliferation of the DPSCs. Low concentration (2% w/v) pectin was biocompatible with the DPSCs and showed an antimicrobial effect against single-species biofilms. This suggests the potential for using pectin as an injectable hydrogel for clinical applications in regenerative endodontics.

BACKGROUND: This study investigated the biocompatibility and antibacterial efficacy of chitosan-gelatin (CH-G) scaffolds loaded with slow-releasing antibiotic formulations used in regeneration endodontic procedures (REPs).
METHODS: Scaffolds were fabricated using freeze drying and loaded with varying concentrations of augmentin or modified triple antibiotic paste (mTAP). High-resolution scanning electron microscopy (SEM) was used to characterize the scaffold, while drug release was monitored via UV-Vis spectrophotometry. Immortalized human mesenchymal stem cells (hMSCs) were cultured on CH-G scaffolds alone (control), either 0.1 mg/mL or 1 mg/mL of augmentin or mTAP, and 10 mg/mL calcium hydroxide (Ca(OH)2). Cell viability and proliferation were assessed using the Alamar Blue assay and SEM, respectively, and live/dead staining further corroborated cell viability. Antibacterial activity against Enterococcus faecalis was evaluated using the MTT assay and confocal laser scanning microscopy (CLSM).
RESULTS: Augmentin at 0.1 mg/mL appeared to promote better cell growth and attachment within the scaffolds than all other formulations, exhibiting acceptable viability. SEM revealed improved cell attachment in augmentin and mTAP groups compared to the Ca(OH)2 group. Augmentin at 1 mg/mL and mTAP groups significantly reduced viable bacteria compared to controls. Augmentin groups and mTAP at 1 mg/mL were highly effective in eliminating E. faecalis biofilms, with mTAP potentially causing more cell death within the remaining biofilm structures.
CONCLUSIONS: This study suggests that CH-G scaffolds loaded with augmentin and mTAP, particularly at a concentration of 1 mg/mL, offer promising advantages for REPs due to their biocompatibility, antibacterial efficacy, and ability to promote cell attachment. Further research may explore the long-term effects in clinical settings.

Background: Antibiotic pastes used as intracanal medication in cases of revascularization therapy might cause negative effects on tooth properties, such as a reduction in dentin microhardness. This in vitro study investigated dentin microhardness in three different locations distancing from the canal lumen after 20 days of treatment with a tri-antibiotic paste (ciprofloxacin, metronidazole, and minocycline), and with a double-antibiotic paste (ciprofloxacin and metronidazole), with calcium hydroxide [Ca(OH)2] UltracalTM XS-treated dentin as comparison. Material and Methods: Human mandibular premolars (n = 48) had the root canals cleaned and shaped and were used to produce dentin slices. Dentin slices remained immersed in the medications for 20 days. The Knoop microhardness (KHN) test was performed before (baseline/Day-0) and after treatment (Day-20) with the medications. Indentations were made at 25 µm, 50 µm, and 100 µm distances from the root canal lumen. The KHN was compared intra-group using Wilcoxon\'s test. Independent groups were compared using Mann-Whitney\'s and Kruskal-Wallis\' tests, at α = 5%. Results: The microhardness in all the tested groups was reduced at Day-20 in comparison with Day-0 (p < 0.001) (intra-group comparison/same distances). The Day-0 values were similar, and the Day-20 values were higher for the Ca(OH)2 group (p < 0.05) (comparison between groups/same distances). Conclusions: Calcium hydroxide for 20 days would be preferred rather than antibiotic pastes to minimize the expected reduction in dentin microhardness during regenerative procedures.

OBJECTIVE: To evaluate histologically and radiographically the potential of dog\'s immature roots with apical periodontitis to regenerate after regenerative endodontic treatment using mesoporous silica nanoparticles (MSNs) with/without bone morphogenic protein (BMP-2) as scaffolds.
METHODS: In 4 mongrel dogs, 56 immature teeth with 96 roots were infected, resulting in necrotic pulps and periapical pathosis. According to the evaluation time (Group I = 30 days and Group II = 90 days), 90 roots were divided into two equal groups (45 roots each) and 6 roots used to replace any lost root during the procedure. The two main groups were further divided according to treatment protocol into 5 subgroups (9 roots each): blood clot (BC subgroup), mesoporous silica nanoparticles scaffold only (MSNs subgroup), mesoporous silica nanoparticles impregnated with BMP2 (MSNs + BMP2 subgroup), infected teeth without treatment (+ ve control subgroup) and normal untouched teeth (-ve control subgroup). All teeth surfaces were coated with Tincture iodine and calcium hydroxide was applied prior to treatment protocols. Then, teeth were restored with glass ionomer filling to seal the remaining part of the access cavity. Radiography evaluation of the increase in root length, root thickness and occurrence of apical closure were performed. Following the sacrifice of the two dogs at each time of evaluation, histopathological analysis was performed and included the inflammatory cells count, bone resorption, tissue ingrowth, deposition of hard tissue, and closure of the apical part. All data were statistically analyzed.
RESULTS: Compared to BC subgroup, MSNs and MSNs + BMP-2 subgroups exhibited significant higher increase in root length and thickness as well as higher vital tissue in-growth and new hard tissue formation in group II (P < 0.05). MSNs + BMP-2 subgroup had significant higher increase in root length and thickness as well as significant lower inflammatory cell count than MSNs subgroup in both groups (P < 0.05). There were no significant differences between MSNs and MSNs + BMP-2 subgroups regarding new hard tissue formation in both groups and apical closure in group I (P > 0.05).
CONCLUSIONS: MSNs with/without BMP-2 scaffolds enabled the continuing growth of roots in immature teeth with necrotic pulps and periapical pathosis. Addition of BMP-2 to MSNs scaffold improved its outcome in regenerative endodontics.
CONCLUSIONS: MSNs with/without BMP-2 scaffolds may alternate blood clot for regenerative endodontic treatment of immature teeth with necrotic pulps.

Dens invaginatus is a developmental dental anomaly that can predispose the tooth to pulp and periradicular disease. Management of this condition can be challenging because of anatomic and microbiologic issues. This case report describes the regenerative endodontic treatment using a strategic antimicrobial protocol for management of an immature maxillary lateral incisor with type-II dens invaginatus associated with apical periodontitis in a 13-year-old patient. The tooth presented with a complex anatomy and was associated with an active sinus tract. Because the true root canal was not negotiable in its coronal part due to the presence of the dens invaginatus, the closed end of the invagination (pseudocanal) was perforated to permit access to the apical segment of the true root canal for cleaning and disinfection. Both the invagination and the true canal were treated using an antimicrobial regimen based on chemomechanical preparation with sodium hypochlorite irrigation, supplementary disinfection with passive ultrasonic irrigation and interappointment calcium hydroxide medication. After 2 exchanges of calcium hydroxide medication, the sinus tract did not disappear, then the antimicrobial protocol was changed to include an antibiotic solution for irrigation and antibiotic paste for intracanal medication. After signs and symptoms disappeared, regenerative endodontic treatment was performed by inducing blood clot formation within the root canal. The coronal canal segment including the invagination was filled with Biodentine. Follow-up including cone-beam computed tomography examination showed complete healing of the apical periodontitis lesion and mineralized tissue formation at the apical portion of the true root canal.

Dentin-pulp regeneration through stem/progenitor cell transplantation represents a promising frontier in regenerative endodontics. This systematic review meticulously evaluates animal studies to investigate the efficacy of stem cell therapy in repairing/regenerating the dentine-pulp complex in mature/immature animal teeth. Employing a comprehensive electronic search of PubMed and Scopus databases up to October 2023, relevant English studies were identified/assessed. Evaluation parameters encompassed radiographic and histological assessments of dentin-pulp complex formation. Outcome measures included pulp-like and dentin-like tissues regeneration, apical healing, dentin thickening, apical closure, and dentinal bridge formation. The risk-of-bias assessment adhered to the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) guidelines. Out of 3250 identified articles, 23 animal experiments were included, categorized into regenerative procedures in mature teeth (n=11), regenerative procedures in immature teeth (n=4), and vital pulp therapy (n=8). Despite the promising potential, the bias in the included studies was high. Notably, Various scaffolds, and growth factors were employed, highlighting the heterogeneity across the studies. Dental pulp stem cells (DPSCs) and bone marrow stem cells, especially specific subfractions, demonstrated notable regenerative potential: hypoxic conditions and extracellular vesicles from preconditioned DPSCs enhanced regeneration, with considerations of cell fate. Donor age impacted regeneration, and challenges persisted in pulpotomy and direct pulp capping. Scaffold and growth factor choices influenced outcomes, underscoring the need for standardized strategies. Despite the promise, clinical viability faces hurdles, necessitating further investigation into adverse effects, optimized scaffolds, and regulatory considerations. This systematic review illuminates the potential of stem cell transplantation for dentin-pulp complex regeneration. The overall evidence quality, influenced by study heterogeneity and biases, underscores the need for cautious interpretation of findings. Future studies should refine methodologies and establish reliable histological parameters for meaningful advancements in dentin-pulp regeneration.