Cytotoxicity, biocompatibility and osteoinductive profile of an MTA-hydrogel-based cement: An in vitro and animal study.

AIM: This study aimed to evaluate the cytotoxicity, biocompatibility and osteoinductive profile of a mineral trioxide aggregate (MTA)-hydrogel-based material (MTA Flow) in comparison with MTA Angelus. METHODOLOGY: Cell viability was evaluated in human periodontal ligament stem cells (hPDLSCs) using the methyl-thiazol-tetrazolium (MTT) colourimetric assay. Polyethylene tubes containing the tested materials and empty polyethylene tubes (control) were implanted in the subcutaneous tissue of Wistar rats. Cellular (lymphocyte infiltration) and extracellular events (ECM; collagen fibres) were analysed in histological sections. Immunohistochemical (collagen I, osteopontin, bone sialoprotein, bone morphogenetic protein4) analyses were also performed. RESULTS: At 24, 48 and 72 h, all tested groups showed cell viability similar to control (p > .05). Regarding biocompatibility, all groups showed similar cellular events represented by a slight inflammatory reaction characterized by hyperaemia and a mild lymphocytic inflammatory infiltrate. The analysis of lymphocytes during the time showed a decrease in these cells in the control group and a significant interaction between MTA Angelus and control (p < .001), with MTA Angelus showing a more extensive inflammatory infiltrate. Regarding fibres, an increase in content was observed in all groups during the experimental time (7, 30 and 60 days), however, no difference was detected among the experimental groups (p = .063). After 60 days, the immunoexpression of bone matrix proteins in the MTA Flow group was similar to or higher than that observed in the MTA Angelus and in the control group. CONCLUSIONS: MTA Flow showed a non-cytotoxic behaviour, biocompatibility and ability to stimulate tissue mineralization.

Cell sheets of human dental pulp stem cells for future application in bone replacement.

OBJECTIVES: To analyze the potential of human dental pulp stem cells (hDPSCs) for maintaining their undifferentiated status and osteogenic differentiation capacity when arranged in cell sheets (CSs) for future application in bone replacement. MATERIALS AND METHODS: CSs were formed after being induced for 10-15 days by clonogenic medium containing additional vitamin C (20 µg/ml). The cell viability of hDPSC4s in the CSs was followed until 96 h using the Live/Dead® assay. The cells of the CSs were enzymatically dissociated and then compared with the original hDPSC4s. The two cell types were characterized immunophenotypically by flow cytometry using specific mesenchymal stem cell-associated markers (CD105, CD146, CD44, STRO-1, and OCT3/4) and non-associated markers (CD34, CD45, and CD14). Osteogenic differentiation was analyzed with the Alizarin red assay. RESULTS: Living cells were observed until 96 h in the CSs. Both cell types exhibited osteogenic differentiation and expressed the specific undifferentiated MSC-associated markers. Cells spontaneously detached from the CSs attached and proliferated at the bottom of the culture dishes. CONCLUSIONS: Cells in the hDPSC4s cell sheets survived for at least 96 h. Moreover, the cells in the cell sheets retained their stemness and their osteogenic differentiation potential. CLINICAL RELEVANCE: Cell sheets of hDPSCs could be employed as natural tri-dimensional structures for treating bone loss. This technique would be useful particularly for critical bone defects or any type of bone defects in patients carrying diseases that impair bone regeneration, such as diabetes mellitus, medication-related osteonecrosis of the jaw (MRONJ), and osteoporosis.

Relationship between toothpastes properties and patient-reported discomfort: crossover study.

OBJECTIVES: This study aims to correlate patient-reported reactions with in vitro analyses of the pH, abrasive quality, and cytotoxicity of four toothpastes. MATERIALS AND METHODS: One hundred twenty-one patients received non-identified samples of toothpaste to be used for 6 days and answered a questionnaire about their sensations. In vitro analysis: the pH of toothpastes was measured with a pH meter. The abrasivity of toothpastes was evaluated against composite resin specimens (n = 10). A toothbrushing machine was used to simulate wear, which was indirectly measured by mass loss using a scale. Cell culture media conditioned with toothpaste were used to assess the cytotoxicity. Confluent cells were kept in contact with the conditioned media or control for 24 h. The cell viability was measured using the 3-(bromide, 4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium (MTT)-reduction assay. The obtained data on the pH, weight loss, and cell viability were compared by ANOVA/Tukey's tests (p < 0.05). RESULTS: With the exception of the bleaching effect paste, the Oral B® paste produced the highest frequencies of irritation reports, tooth sensitivity, taste discomfort, and texture discomfort in the clinical study; patients also reported rougher teeth, soft tissue peeling, dry mouth, thrush, tingling, and taste changes in response to this paste. The in vitro analysis demonstrated that Oral B® had the lowest pH, the highest abrasivity, and produced the lowest cell viability (p < 0.01). CONCLUSION: Results suggest that low pH toothpastes that are highly abrasive and cytotoxic may cause undesirable reactions in patients. CLINICAL RELEVANCE: Toothpaste's properties should be well known for indication to patient therefore minimizing discomfort reports.

Evaluation of pain, jaw movements, and psychosocial factors in elderly individuals with temporomandibular disorder under laser phototherapy.

Few studies have been carried out on the application of laser phototherapy (LPT) for treating painful temporomandibular disorder (TMD) in elderly population that is growing worldwide. The aim of the present study was to evaluate the pain, jaw movements, and psychosocial factors in ten elderly patients with painful TMD before and after LPT. All patients were evaluated before and after LPT by using the Research Diagnostic Criteria for temporomandibular disorders (RDC/TMD) axes I and II. For pain assessment, a visual analogue scale (VAS) was used. The LPT was carried out with an GaAlAs diode laser (780 nm; spot size 0.04 cm(2)) in punctual and contact mode. Two settings of irradiations were applied as follows: in patients presenting myofascial pain, 10 mW, 5 J/cm(2), 20 s, 0.2 J per application point; and in patients with joint TMD, 70 mW, 105 J/cm(2), 60 s on five points, 4.2 J per point. Two sessions of LPT were carried out per week over four consecutive weeks, in the total of eight sessions. Data was statistically analyzed (p < 0.05). Significant pain reduction was found in all patients. There were increase in maximum mouth opening without pain and reduction in muscle pain during right and left lateral excursion. A significant reduction in chronic pain severity (p = 0.02) and significant improvements in depression (p = 0.038) and nonspecific physical symptoms with pain (p = 0.0167) were observed. The present findings indicate that LPT is able to promote pain relief and improvement of jaw movements in elderly patients with TMD, with a positive effect on psychosocial aspects.

Influence of CO2 (10.6 µm) and Nd:YAG laser irradiation on the prevention of enamel caries around orthodontic brackets.

One possible undesirable consequence of orthodontic therapy is the development of incipient caries lesions of enamel around brackets. The aim of this study was to compare the effects of CO2 (λ = 10.6 µm) and Nd:YAG (λ = 1,064 nm) lasers associated or not with topical fluoride application on the prevention of caries lesions around brackets. Brackets were bonded to the enamel of 65 premolars. The experimental groups (n = 13) were: G1--application of 1.23% acidulated fluoride phosphate gel (AFP, control); G2--Nd:YAG laser irradiation (0.6 W, 84.9 J/cm(2), 10 Hz, 110 µs, contact mode); G3--Nd:YAG laser irradiation associated with AFP; G4--CO2 laser irradiation (0.5 W, 28.6 J/cm(2), 50 Hz, 5 µs, and 10 mm focal distance); and G5--CO2 laser irradiation associated with AFP. Quantitative light-induced fluorescence was used to assess enamel demineralization. The data were statistically compared (α = 5%). The highest demineralization occurred in the Nd:YAG laser group (G2, 26.15% ± 1.94). The demineralization of all other groups was similar to that of the control group. In conclusion, CO2 laser alone was able to control enamel demineralization around brackets at the same level as that obtained with topical fluoride application.

New uses for rehabilitation protocol for oral sinus communications in ARONJ patients.

This report presents a new use for rehabilitation protocol for oral sinus communications in patients with antiresorptive agent-induced osteonecrosis of the jaw. The treatment plan consisted of constructing an atraumatic complete denture with rounded edges, made with nontoxic resin, to prevent any injury to the mucosa and recurrence of the disease. The patient was followed up for 4 years, without any complications, and was socially reintegrated by resuming the normal life he experienced before tooth loss.

Osteoradionecrosis in a Torus Mandibularis Treated by Antimicrobial Photodynamic Therapy: A Case Report.

Background: Osteoradionecrosis (ORN) of the jaws is a late complication after radiotherapy to head and neck cancer. Objective: To describe a rare case of ORN of the torus mandibularis that was successfully managed exclusively with antimicrobial photodynamic therapy (aPDT). Case report: A 72-year-old man presented an exposed necrotic bone observed in the torus mandibularis, extending to the lingual alveolar ridge with no edema nor suppuration. The treatment provided a noninvasive treatment leading to spontaneous sequestrectomy of the torus in 2 weeks with complete mucosal repair in 5 weeks and absence of lesion signs and/or symptoms even after 6 months of follow-up. Conclusions: The aPDT indicated to be a satisfactory treatment for ORN affecting torus mandibularis, a region with surgical limitations, avoiding surgery.

Gingival cyst of the adult.

The gingival cyst of the adult (GCA) is a rare odontogenic cyst, consisting of 0.3% of all odontogenic cysts. This case report, based on CARE guidelines for case reports, aims to present a case of a 52-year-old female patient with a symptomatic translucent nodule in the upper left anterior gingiva, measuring approximately 6mm. Excisional biopsy was performed, and the histological examination revealed multiple cystic cavities lined by the squamous epithelium of varying thickness with focal areas of nodular thickenings. The presence of clusters of cells with clear cytoplasm within epithelial thickenings was observed. PAS staining was negative in clear cells. The diagnosis of the GCA was established. Despite its rarity, GCA should be considered in the differential diagnosis of gingival lesions. Conservative surgical treatment proved to be effective, with no signs of recurrence.

Genotoxicity and Cytotoxicity Comparison of Calcium Silicate-Based and Resin-Based Sealers on Human Periodontal Ligament Stem Cells.

OBJECTIVE: This study aimed to assess the genotoxicity and cytotoxicity of Sealer Plus BC (SBC), AH Plus (AHP) and MTA Fillapex (MTF). METHODS: Human periodontal ligament dental stem cells (hPDLSCs) from third molars were isolated and cultured in a clonogenic medium. Cells were maintained in an incubator, and cell growth was monitored daily. hPDLSCs were characterised under flow cytometry and stem cell surface markers. The tested groups were a control group, SBC, AHP and MTF. Each sealer was prepared according to the manufacturer's instructions and placed in a clonogenic medium to produce a conditioned media. Conditioned media were then diluted to 10% to be placed in contact with culture cells in cell viability assay afterwards. The cells were harvested and plated into 96 wells culture plates. Genotoxicity was assessed by evaluation of micronucleus formation and cytotoxicity by MTT-based assay. All experiments were performed in triplicate. Data normality was verified by the Kolmogorov-Smirnov test. Statistical analysis for genotoxicity was performed with Kruskal-Wallis and Dunn's tests and two-way ANOVA for cytotoxicity, both with a significance level of 5%. RESULTS: Cells expressed typical levels of mesenchymal stem cell surface markers. No differences in the number of micronuclei were observed among all groups (P>0.05). In all periods analysed (24, 48, and 72 h), the sealers presented statistically different results for cell viability (P<0.05), with SBC presenting the lowest cytotoxicity, followed by the control group, MTF, and AHP. CONCLUSION: All sealers presented low genotoxicity, and Sealer Plus BC presented the lowest cytotoxicity.

Pro-angiogenic potential of a functionalized hydrogel scaffold as a secretome delivery platform: An innovative strategy for cell homing-based dental pulp tissue engineering.

Angiogenesis is a key process that provides a suitable environment for successful tissue engineering and is even more crucial in regenerative endodontic procedures, since the root canal anatomy limits the development of a vascular network supply. Thus, sustainable and accelerated vascularization of tissue-engineered dental pulp constructs remains a major challenge in cell homing approaches. This study aimed to functionalize a chitosan hydrogel scaffold (CS) as a platform loaded with secretomes of stem cells from human exfoliated deciduous teeth (SHEDs) and evaluate its bioactive function and pro-angiogenic properties. Initially, the CS was loaded with SHED secretomes (CS-S), and the release kinetics of several trophic factors were assessed. Proliferation and chemotaxis assays were performed to analyze the effect of functionalized scaffold on stem cells from apical papilla (SCAPs) and the angiogenic potential was analyzed through the Matrigel tube formation assay with co-cultured of human umbilical vein endothelial cells and SCAPs. SHEDs and SCAPs expressed typical levels of mesenchymal stem cell surface markers. CS-S was able to release the trophic factors in a sustained manner, but each factor has its own release kinetics. The CS-S group showed a significantly higher proliferation rate, accelerated the chemotaxis, and higher capacity to form vascular-like structures. CS-S provided a sustained and controlled release of trophic factors, which, in turn, improved proliferation, chemotaxis and all angiogenesis parameters in the co-culture. Thus, the functionalization of chitosan scaffolds loaded with secretomes is a promising platform for cell homing-based tissue engineering.

Effects of the crosslinking of chitosan/DCPA particles in the antimicrobial and mechanical properties of dental restorative composites.

The development of restorative materials containing antibacterial agents is an alternative to reduce the progression of caries lesions. OBJECTIVE: to compare the influence of the degree of crosslinking of chitosan particles loaded with dibasic calcium phosphate (DCPA) on the mechanical properties, degree of conversion (DC), and antimicrobial properties of experimental composites. METHODS: Chitosan/DCPA particles were synthesized by the electrospraying, crosslinked by 0, 8, or 16 h in glutaraldehyde, and characterized by zeta potential and minimum inhibitory concentration (MIC) against S. mutans. Experimental resin composites of Bis-GMA and TEGDMA and 59.5% of barium glass were synthesized, chitosan/DCPA particles were added at 0 or 0.5 wt% with the different crosslinking time. The materials were subject to DC analysis, three-point bending test at 24 h and 7 days, and antimicrobial assays. Data were submitted to one-way ANOVA and Tukey test (α = 0.05). RESULTS: The particles with longer crosslinking time presented higher zeta potential and MIC, and the composite containing these particles showed significantly higher biofilm inhibition than the control group. The other two groups were similar to each other and the control. The composite containing particles with 88 h crosslinking time showed the lowest flexural strength at 7 days in water, and materials with non-crosslinked particles and longer crosslinking time presented flexural strength similar to control. The flexural modulus and DC showed no statistical difference among groups. SIGNIFICANCE: composite resin containing 0.5% chitosan/DCPA particles crosslinked by 16 h showed a reduction of biofilm formation without affecting the mechanical properties in relation to the control.

Dental tissue-derived stem cell sheet biotechnology for periodontal tissue regeneration: A systematic review.

OBJECTIVE: This study aimed to conduct a systematic review of the use of a cell sheet formed by mesenchymal stem cells derived from dental tissues (ddMSCs) for periodontal tissue regeneration in animal models in comparison with any other type of regenerative treatment. DESIGN: PubMed and Scopus databases were searched for relevant studies up to December 2020. The review was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. RESULTS: Of the 1542 potentially relevant articles initially identified, 33 fulfilled the eligibility criteria and were considered for this review. Even with a wide variety of selected study methods, the periodontal tissue was always regenerated; this indicates the potential for the use of these cell sheets in the future of periodontics. However, this regeneration process is not always complete. CONCLUSION: Despite the implantation, ddMSCs sheets have a great potential to be used in the regeneration of periodontal tissue. More in vivo studies should be conducted using standardized techniques for cell sheet implantation to obtain more robust evidence of the relevance of using this modality of cell therapy for periodontal tissue regeneration.

Effect of human dental pulp stem cell conditioned medium in the dentin-pulp complex regeneration: A pilot in vivo study.

BACKGROUND: Dental trauma, restorative operative procedures and/or caries lesions can expose the dental pulp. Facing this clinical condition, where the maintenance of the dentin-pulp complex vitality is imperative, is challenging in Dentistry. Dental pulp stem cells conditioned medium contains trophic factors that could help in this task. This in vivo pilot study aimed to evaluate the effects of the human dental pulp stem cells conditioned medium on the dental pulp tissue response to vital pulp therapy. MATERIAL AND METHODS: Concentrated conditioned medium was obtained by incubating characterized human dental pulp stem cells with fresh culture medium. Pulp exposures performed at the first upper molars (n = 20) of Wistar rats were directly capped with: MTA or MTA + Conditioned Medium. Four and 8 weeks later, the samples were qualitatively analyzed in histological sections (H&E). RESULTS: When the conditioned medium was associated with MTA, there were a high percentage of samples presenting formation of dentin bridges and small percentage of pulp tissue with inflammatory signs in both experimental times. The conditioned medium improved the organization of the newly formed hard tissue. CONCLUSIONS: The association of dental pulp stem cell conditioned medium with MTA showed beneficial effects on dentin-pulp complex regeneration and has promising potential for studies in regenerative dentistry.

Development and characterization of a new chitosan-based scaffold associated with gelatin, microparticulate dentin and genipin for endodontic regeneration.

OBJECTIVE: An ideal scaffold for endodontic regeneration should allow the predictableness of the new tissue organization and limit the negative impact of residual bacteria. Therefore, composition and functionalization of the scaffold play an important role in tissue bioengineering. The objective of this study was to assess the morphological, physicochemical, biological and antimicrobial properties of a new solid chitosan-based scaffold associated with gelatin, microparticulate dentin and genipin. METHODS: Scaffolds based on chitosan (Ch); chitosan associated with gelatin and genipin (ChGG); and chitosan associated with gelatin, microparticulate dentin and genipin (ChGDG) were prepared by using the freeze-drying method. The morphology of the scaffolds was analyzed by scanning electron microscopy (SEM). The physicochemical properties were assessed for biodegradation, swelling and total released proteins. The biological aspects of the scaffolds were assessed using human cells from the apical papilla (hCAPs). Cell morphology and adhesion to the scaffolds were evaluated by SEM, cytotoxicity and cell proliferation by MTT reduction-assay. Cell differentiation in scaffolds was assessed by using alizarin red assay. The antimicrobial effect of the scaffolds was evaluated by using the bacterial culture method, and bacterial adhesion to the scaffolds was observed by SEM. RESULTS: All the scaffolds presented porous structures. The ChCDG had more protein release, adhesion, proliferation and differentiation of hCAPs, and bacteriostatic effect on Enterococcus faecalis than Ch and ChGG (p < 0.05). SIGNIFICANCE: The chitosan associated with gelatin, microparticulate dentin and genipin has morphological, physicochemical, biological and antibacterial characteristics suitable for their potential use as scaffold in regenerative endodontics.

Biological Properties of Experimental Methacrylate-Based Sealers Containing Calcium Phosphates.

This study aimed to evaluate, in vitro and in vivo, the biocompatibility of experimental methacrylate-based endodontic sealers containing α-tricalcium phosphate (α-TCP) or nanostructured hydroxyapatite (HAp). Experimental methacrylate-based dual-cure sealers with the addition of α-TCP or HAp, at 10%wt were formulated and compared to AH Plus (AHP). Cell viability was assessed by 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT), and sulforhodamine B (SRB). Sealers were implanted in rats' subcutaneous tissue and histologically evaluated. Bioactivity was assessed by alkaline phosphatase enzyme activity (ALP) and Alizarin Red (AR), using apical papillary cells (SCAPs), and by the bone deposition measured in surgical cavities on rats' femur filled with AH Plus or α-TCP. In both viability assays, HAp and AHP sealers were similar, and α-TCP presented lower viability compared to the others at MTT assay (p<0.05). A gradual decrease of the inflammatory response according to the periods was observed and AHP was the only that presented giant cells (7-day period). Collagen fibers condensation increased according to the periods, with no differences among sealers. There was an increase at ALP activity and mineralized nodules deposition according to periods. HAp and α-TCP presented higher values for ALP activity at 5 days and at 5, 10, and 15 days for AR and were different from AHP (p<0.05). α-TCP presented superior values at 10 and 15 days compared to HAp and AHP for AR (p<0.05). At 90 days, α-TCP and control (empty cavity) showed high bone deposition compared to AHP (p<0.05). α-TCP and HAp, in a methacrylate-based sealer, presented biocompatibility and bioactivity, with the potential to be used as endodontic sealers in clinical practice. Further investigations are required to gain information on the physicochemical properties of these sealers formulation before its clinical implementation.

Physical and Biological Properties of a Chitosan Hydrogel Scaffold Associated to Photobiomodulation Therapy for Dental Pulp Regeneration: An In Vitro and In Vivo Study.

BACKGROUND: The regeneration of dental pulp, especially in cases of pulp death of immature teeth, is the goal of the regenerative endodontic procedures (REPs) that are based on tissue engineering principles, consisting of stem cells, growth factors, and scaffolds. Photobiomodulation therapy (PBMT) showed to improve dental pulp regeneration through cell homing approaches in preclinical studies and has been proposed as the fourth element of tissue engineering. However, when a blood clot was used as a scaffold in one of these previous studies, only 30% of success was achieved. The authors pointed out the instability of the blood clot as the regeneration shortcoming. Then, to circumvent this problem, a new scaffold was developed to be applied with the blood clot. The hypothesis of the present study was that an experimental injectable chitosan hydrogel would facilitate the three-dimensional spatial organization of endogenous stem cells in dental pulp regeneration with no interference on the positive influence of PBMT. METHODS: For the in vitro analysis, stem cells from the apical papilla (SCAPs) were characterized by flow cytometry and applied in the chitosan scaffold for evaluating adhesion, migration, and proliferation. For the in vivo analysis, the chitosan scaffold was applied in a rodent orthotopic dental pulp regeneration model under the influence of PBMT (660 nm; power output of 20 mW, beam area of 0.028 cm2, and energy density of 5 J/cm2). RESULTS: The scaffold tested in this study allowed significantly higher viability, proliferation, and migration of SCAPs in vitro when PBMT was applied, especially with the energy density of 5 J/cm2. These results were in consonance to those of the in vivo data, where pulp-like tissue formation was observed inside the root canal. CONCLUSION: Chitosan hydrogel when applied with a blood clot and PBMT could in the future improve previous results of dental pulp regeneration through cell homing approaches.

Human Stem Cells and Tissue Responses to Xenogeneic Bone Block: An In Vitro and Histologic Study.

PURPOSE: Premature loss of a tooth element causes extensive local bone atrophy. Block biomaterials are used in patients with an atrophic maxilla to reshape the premaxilla bone. This study aimed to evaluate the responses to xenogeneic bone blocks at cellular and tissue levels. MATERIALS AND METHODS: Viability of human periodontal ligament stem cells (hPDLSCs) submitted to culture medium-containing substances leached from a xenogeneic block biomaterial (Orthogen) was assessed by the MTT assay. Cells grown in a fresh medium were used as the control. Cell viability was measured at 24, 48, and 72 hours. The results were statistically compared (α ≤ .05). Tissue response to the biomaterial was evaluated by the placement of xenogeneic blocks anchored with miniscrews in six patients with atrophic maxillae and a remaining cortical bone tissue thickness of 2.0 to 3.0 mm. After 8 months, specimens were collected from the grafted areas and analyzed under a light microscope for evaluating the types of collagen fibers and amount of newly formed bone tissue, using the Image-Pro Plus software program. RESULTS: The number of viable cells was similar in both groups. The histologic sections revealed remaining biomaterial in 10.12% of the patients and newly formed bone in 21.57%. CONCLUSION: The xenogeneic block biomaterial tested proved to be biocompatible in vitro, and the porosity of the block favored the in vivo formation of new bone matrix deposits.

Current evidence of tissue engineering for dentine regeneration in animal models: a systematic review.

Aim: The aim of this study is to verify the type of scaffold effect on tissue engineering for dentine regeneration in animal models. Materials & methods: Strategic searches were conducted through MEDLINE/PubMed, Web of Science and Scopus databases. The studies were included with the following eligibility criteria: studies evaluating dentine regeneration, and being an in vivo study. Results: From 1392 identified potentially relevant studies, 15 fulfilled the eligibility criteria. All studies described characteristics of neoformed dentine, being that the most reported reparative dentine formation. Most of included studies presented moderate risk of bias. Conclusion: Up to date scientific evidence shows a positive trend to dentine regeneration when considering tissue engineering in animal models, regardless the type of scaffolds used.

Development of novel dental restorative composites with dibasic calcium phosphate loaded chitosan fillers.

The incorporation of antimicrobial agents in restorative dental composites has the potential to slow the development of carious lesions. OBJECTIVE: The objectives of the present study were to develop experimental composite resins with chitosan or chitosan loaded with dibasic calcium phosphate anhydrous (DCPA) particles and to demonstrate their antimicrobial potential without loss of mechanical properties or biocompatibility. METHODS: Chitosan and chitosan/DCPA particles were synthetized by the electrospray method. Experimental composites were formulated by adding 0, 0.5, or 1.0 wt% particles into a resin matrix along with 60 wt% barium glass. The degree of conversion and mechanical properties were measured after 1 and 90 days of aging in water after photoactivation. Cytotoxicity and genotoxicity were evaluated using fibroblasts from dental pulp in conditioned medium. The antimicrobial activity against Streptococcus mutans was assessed by crystal violet biofilm assay. RESULTS: The experimental restorative composites were not found to be cytotoxic or genotoxic, with cell viability of 93.1 ± 8.0% (p = 0.328) and 3.0 ± 0.8% micronucleus per group (p = 0.1078), respectively. The antimicrobial results showed that all composites with approximately 20% less biofilm (p < 0.001) relative to the control. No chitosan release was detected from the composites, suggesting direct contact of the bacteria with exposed chitosan particles on the surface was responsible for the observed antimicrobial effect. The addition of the chitosan and chitosan/DCPA submicrometer (<250 nm average diameter) particles to restorative composites did not change the degree of conversion, flexural strength, elastic modulus and fracture toughness compared to the control group after 90 days aging in water. SIGNIFICANCE: It can be concluded that the addition of chitosan or chitosan/DCPA particles in the restorative composites induced antimicrobial activity without compromising the mechanical properties or biocompatibility of the composites.