Bioprinting PDLSC-Laden Collagen Scaffolds for Periodontal Ligament Regeneration.

Periodontitis and severe trauma are major causes of damage to the periodontal ligament (PDL). Repairing the native conditions of the PDL is essential for the stability of the tissue and its interfaces. Bioprinting periodontal ligament stem cells (PDLSCs) is an interesting approach to guide the regeneration of PDL and interfacial integration. Herein, a collagen-based bioink mimicking the native extracellular matrix conditions and carrying PDLSCs was tested to guide the periodontal ligament organization. The bioink was tested at two different concentrations (10 and 15 mg/mL) and characterized by swelling and degradation, microstructural organization, and rheological properties. The biological properties were assessed after loading PDLSCs into bioinks for bioprinting. The characterization was performed through cell viability, alizarin red assay, and expression for ALP, COL1A1, RUNX2, and OCN. The in vivo biocompatibility of the PDLSC-laden bioinks was verified using subcutaneous implantation in mice. Later, the ability of the bioprinted PDLSC-laden bioinks on dental root fragments to form PDL was also investigated in vivo in mice for 4 and 10 weeks. The bioinks demonstrated typical shear-thinning behavior, a porous microstructure, and stable swelling and degradation characteristics. Both concentrations were printable and provided suitable conditions for a high cell survival, proliferation, and differentiation. PDLSC-laden bioinks demonstrated biocompatibility in vivo, and the bioprinted scaffolds on the root surface evidenced PDLSC alignment, organization, and PDLSC migration to the root surface. The versatility of collagen-based bioinks provides native ECM conditions for PDLSC proliferation, alignment, organization, and differentiation, with translational applications in bioprinting scaffolds for PDL regeneration.

Recent advances in additive manufacturing of patient-specific devices for dental and maxillofacial rehabilitation.

OBJECTIVES: Customization and the production of patient-specific devices, tailoring the unique anatomy of each patient's jaw and facial structures, are the new frontiers in dentistry and maxillofacial surgery. As a technological advancement, additive manufacturing has been applied to produce customized objects based on 3D computerized models. Therefore, this paper presents advances in additive manufacturing strategies for patient-specific devices in diverse dental specialties. METHODS: This paper overviews current 3D printing techniques to fabricate dental and maxillofacial devices. Then, the most recent literature (2018-2023) available in scientific databases reporting advances in 3D-printed patient-specific devices for dental and maxillofacial applications is critically discussed, focusing on the major outcomes, material-related details, and potential clinical advantages. RESULTS: The recent application of 3D-printed customized devices in oral prosthodontics, implantology and maxillofacial surgery, periodontics, orthodontics, and endodontics are presented. Moreover, the potential application of 4D printing as an advanced manufacturing technology and the challenges and future perspectives for additive manufacturing in the dental and maxillofacial area are reported. SIGNIFICANCE: Additive manufacturing techniques have been designed to benefit several areas of dentistry, and the technologies, materials, and devices continue to be optimized. Image-based and accurately printed patient-specific devices to replace, repair, and regenerate dental and maxillofacial structures hold significant potential to maximize the standard of care in dentistry.

Unveiling the potential of melt electrowriting in regenerative dental medicine.

For nearly three decades, tissue engineering strategies have been leveraged to devise effective therapeutics for dental, oral, and craniofacial (DOC) regenerative medicine and treat permanent deformities caused by many debilitating health conditions. In this regard, additive manufacturing (AM) allows the fabrication of personalized scaffolds that have the potential to recapitulate native tissue morphology and biomechanics through the utilization of several 3D printing techniques. Among these, melt electrowriting (MEW) is a versatile direct electrowriting process that permits the development of well-organized fibrous constructs with fiber resolutions ranging from micron to nanoscale. Indeed, MEW offers great prospects for the fabrication of scaffolds mimicking tissue specificity, healthy and pathophysiological microenvironments, personalized multi-scale transitions, and functional interfaces for tissue regeneration in medicine and dentistry. Excitingly, recent work has demonstrated the potential of converging MEW with other AM technologies and/or cell-laden scaffold fabrication (bioprinting) as a favorable route to overcome some of the limitations of MEW for DOC tissue regeneration. In particular, such convergency fabrication strategy has opened great promise in terms of supporting multi-tissue compartmentalization and predetermined cell commitment. In this review, we offer a critical appraisal on the latest advances in MEW and its convergence with other biofabrication technologies for DOC tissue regeneration. We first present the engineering principles of MEW and the most relevant design aspects for transition from flat to more anatomically relevant 3D structures while printing highly-ordered constructs. Secondly, we provide a thorough assessment of contemporary achievements using MEW scaffolds to study and guide soft and hard tissue regeneration, and draw a parallel on how to extrapolate proven concepts for applications in DOC tissue regeneration. Finally, we offer a combined engineering/clinical perspective on the fabrication of hierarchically organized MEW scaffold architectures and the future translational potential of site-specific, single-step scaffold fabrication to address tissue and tissue interfaces in dental, oral, and craniofacial regenerative medicine. STATEMENT OF SIGNIFICANCE: Melt electrowriting (MEW) techniques can further replicate the complexity of native tissues and could be the foundation for novel personalized (defect-specific) and tissue-specific clinical approaches in regenerative dental medicine. This work presents a unique perspective on how MEW has been translated towards the application of highly-ordered personalized multi-scale and functional interfaces for tissue regeneration, targeting the transition from flat to anatomically-relevant three-dimensional structures. Furthermore, we address the value of convergence of biofabrication technologies to overcome the traditional manufacturing limitations provided by multi-tissue complexity. Taken together, this work offers abundant engineering and clinical perspectives on the fabrication of hierarchically MEW architectures aiming towards site-specific implants to address complex tissue damage in regenerative dental medicine.

Natural monoterpenes-laden electrospun fibrous scaffolds for endodontic infection eradication.

This investigation aimed to synthesize poly(D,L-lactide) (PLA)-based fibrous scaffolds containing natural essential oils (i.e., linalool and citral) and determine their antimicrobial properties and cytocompatibility as a clinically viable cell-friendly disinfection strategy for regenerative endodontics. PLA-based fibrous scaffolds were fabricated via electrospinning with different concentrations of linalool and citral. The micromorphology and average diameter of the fibers was investigated through scanning electron microscopy (SEM). The chemical composition of the scaffolds was inferred by Fourier-transform infrared spectroscopy (FTIR). Antimicrobial efficacy against Enterococcus faecalis and Actinomyces naeslundii was also evaluated by agar diffusion and colony-forming units (CFU) assays. The scaffolds' cytocompatibility was determined using dental pulp stem cells (DPSCs). Statistical analyses were performed and the significance level was set at α = 5%. Linalool and citral's incorporation in the PLA fibrous scaffolds was confirmed in the FTIR spectra. SEM images indicate no morphological changes upon inclusion of the essential oils, except the reduced diameter of 40% linalool-laden fibers (p < 0.05). Importantly, significant antimicrobial properties were reported for citral-containing scaffolds for CFU/mL counts (p < 0.05), while only 20% and 40% linalool-laden scaffolds reduced CFU/mL (p < 0.05). Meanwhile, the inhibition halos were verified in a concentration-dependent manner for all monoterpenes-laden scaffolds. Citral- and linalool-laden PLA-based fibrous scaffolds showed acceptable cytocompatibility. The incorporation of natural monoterpenes did not alter the scaffolds' fibrous morphology, promoted antimicrobial action against endodontic pathogens, and preserved DPSCs viability. Linalool- and citral-laden electrospun scaffolds hold promise as naturally derived antimicrobial therapeutics for applications in regenerative endodontics.

Self-assembling peptide-laden electrospun scaffolds for guided mineralized tissue regeneration.

OBJECTIVES: Electrospun scaffolds are a versatile biomaterial platform to mimic fibrillar structure of native tissues extracellular matrix, and facilitate the incorporation of biomolecules for regenerative therapies. Self-assembling peptide P11-4 has emerged as a promising strategy to induce mineralization; however, P11-4 application has been mostly addressed for early caries lesions repair on dental enamel. Here, to investigate P11-4's efficacy on bone regeneration, polymeric electrospun scaffolds were developed, and then distinct concentrations of P11-4 were physically adsorbed on the scaffolds. METHODS: P11-4-laden and pristine (P11-4-free) electrospun scaffolds were immersed in simulated body fluid and mineral precipitation identified by SEM. Functional groups and crystalline phases were analyzed by FTIR and XRD, respectively. Cytocompatibility, mineralization, and gene expression assays were conducted using stem cells from human exfoliated deciduous teeth. To investigate P11-4-laden scaffolds potential to induce in vivo mineralization, an established rat calvaria critical-size defect model was used. RESULTS: We successfully synthesized nanofibrous (∼ 500 nm fiber diameter) scaffolds and observed that functionalization with P11-4 did not affect the fibers' diameter. SEM images indicated mineral precipitation, while FTIR and XRD confirmed apatite-like formation and crystallization for P11-4-laden scaffolds. In addition, P11-4-laden scaffolds were cytocompatible, highly stimulated cell-mediated mineral deposition, and upregulated the expression of mineralization-related genes compared to pristine scaffolds. P11-4-laden scaffolds led to enhanced in vivo bone regeneration after 8 weeks compared to pristine PCL. SIGNIFICANCE: Electrospun scaffolds functionalized with P11-4 are a promising strategy for inducing mineralized tissues regeneration in the craniomaxillofacial complex.

Effect of a Self-Assembly Peptide on Surface Roughness and Hardness of Bleached Enamel.

After bleaching, enamel surfaces are damaged, contributing to erosion and tooth sensitivity. Although fluoride is used after bleaching to try and revert alterations, it is not capable of repairing tooth structure. This study compared the effect of a self-assembly peptide (P11-4), with and without fluoride, and sodium fluoride (NaF 2%) on the Knoop microhardness (KHN) and surface roughness (Ra (µm)) of bleached enamel with an in-office bleaching regimen. Enamel blocks of bovine teeth (5 × 5 × 2 mm) with standardized surface hardness were bleached with 35% carbamide peroxide, following the manufacturer's instructions. The teeth were randomly divided into the following groups (n = 7) according to post-bleaching treatment: no treatment (negative control) (C-); 2% NaF (NaF); Curodont™ Repair (Repair); and Curodont™ Protect (Protect). Specimens were stored in artificial saliva at 37 °C. To evaluate the effect of the post-bleaching treatments, KHN and Ra were measured before bleaching (baseline) and 24 h and 7 days after bleaching. Data were submitted to repeated measures ANOVA and Bonferroni tests (α = 0.05). There were significant interactions between the study factors (p = 0.001). After 7 days, Repair (572.50 ± 79.04) and Protect (583.00 ± 74.76) specimens showed increased surface KHN, with values higher than the NaF (465.50 ± 41.50) and C- (475.22 ± 58.95) baseline values. There was no significant difference in KHN at 24 h among groups (p = 0.587). At 24 h after bleaching, Repair was significantly different from all groups (p < 0.05). Repair showed the lowest Ra (µm) values (0.133 ± 0.035). After seven days, there was no significant difference in Ra values among groups when compared to the baseline. The use of P11-4-based materials after bleaching resulted in the fastest recovery to baseline enamel properties.

Innovations in Craniofacial Bone and Periodontal Tissue Engineering - From Electrospinning to Converged Biofabrication.

From a materials perspective, the pillars for the development of clinically translatable scaffold-based strategies for craniomaxillofacial (CMF) bone and periodontal regeneration have included electrospinning and 3D printing (biofabrication) technologies. Here, we offer a detailed analysis of the latest innovations in 3D (bio)printing strategies for CMF bone and periodontal regeneration and provide future directions envisioning the development of advanced 3D architectures for successful clinical translation. First, the principles of electrospinning applied to the generation of biodegradable scaffolds are discussed. Next, we present on extrusion-based 3D printing technologies with a focus on creating scaffolds with improved regenerative capacity. In addition, we offer a critical appraisal on 3D (bio)printing and multitechnology convergence to enable the reconstruction of CMF bones and periodontal tissues. As a future outlook, we highlight future directions associated with the utilization of complementary biomaterials and (bio)fabrication technologies for effective translation of personalized and functional scaffolds into the clinics.

A Highly Ordered, Nanostructured Fluorinated CaP-Coated Melt Electrowritten Scaffold for Periodontal Tissue Regeneration.

Periodontitis is a chronic inflammatory, bacteria-triggered disorder affecting nearly half of American adults. Although some level of tissue regeneration is realized, its low success in complex cases demands superior strategies to amplify regenerative capacity. Herein, highly ordered scaffolds are engineered via Melt ElectroWriting (MEW), and the effects of strand spacing, as well as the presence of a nanostructured fluorinated calcium phosphate (F/CaP) coating on the adhesion/proliferation, and osteogenic differentiation of human-derived periodontal ligament stem cells, are investigated. Upon initial cell-scaffold interaction screening aimed at defining the most suitable design, MEW poly(ε-caprolactone) scaffolds with 500 µm strand spacing are chosen. Following an alkali treatment, scaffolds are immersed in a pre-established solution to allow for coating formation. The presence of a nanostructured F/CaP coating leads to a marked upregulation of osteogenic genes and attenuated bacterial growth. In vivo findings confirm that the F/CaP-coated scaffolds are biocompatible and lead to periodontal regeneration when implanted in a rat mandibular periodontal fenestration defect model. In aggregate, it is considered that this work can contribute to the development of personalized scaffolds capable of enabling tissue-specific differentiation of progenitor cells, and thus guide simultaneous and coordinated regeneration of soft and hard periodontal tissues, while providing antimicrobial protection.

Glass-ionomer cement modifies the gene expression of Streptococcus mutans providing a lower virulent biofilm.

PURPOSE: To evaluate the effect of glass-ionomer cement (GIC) on gene expression (gtfC, gtfD, covR, and vicR) of Streptococcus mutans (S. mutans) biofilms at 2, 4 and 24 hours. METHODS: Six groups were tested according to the materials and time observation, as follows: ceramic (IPS Empress Esthetic), as the control group, and GIC (Ketac Molar Easymix); and time points of S. mutans biofilm formation (2, 4, and 24 hours). Round-shaped samples (10 x 2 mm) of each material were prepared according to the manufacturers' specifications. GIC discs were handled in a laminar flow hood under aseptic conditions and stored at 100% relative humidity at 37°C for 24 hours to complete setting reaction. The samples were placed in a 24-well plate and immersed in 1.5 ml BHI + 1% sucrose with an inoculum of S. mutans UA159 to allow biofilm growth during 2, 4, and 24 hours. Next, the samples were removed, vortexed and centrifuged to collect cell pellets (n=5) for each material and time point. Pellets were stored at -80°C. Then, RNA was purified using the RNeasy Mini Kit protocol. The RNA was converted in cDNA using iScript cDNA Synthesis according to the manufacturer's recommendations. Analysis of gtfC, gtfD, vicR, and covR expressions was performed using Step One Real-Time qPCR device with specific primers for each gene and the analysis normalized by 16S reference gene expression. Data from gtfC, gtfD, and vicR were analyzed by t-test to compare between groups while Mann-Whitney was used to analyze covR expression (α= 0.05). RESULTS: No significant differences at 2 and 4 hours between materials for all analyzed genes were noted. However, in the 24-hour period, a significant decrease in gtfC and vicR expressions were observed, while covR expression increased when GIC was compared to ceramic. CLINICAL SIGNIFICANCE: The use of glass-ionomer cement decreased the virulence of S. mutans biofilms, which may imply a reduced bacterial cariogenic potential.