Expanding the Surgeon's Armamentarium: Use of the Tubing Technique to Preserve the Inferior Alveolar Nerve During Transposition Procedure.

Severe edentulous posterior mandible atrophy with inadequate bone height superior to the inferior alveolar canal may increase the risk of neurosensory impairment and other complications during inferior alveolar nerve (IAN) transposition (IANT) prior to dental implant insertion. The current report describes the tubing technique as a practical and feasible procedure that ensures IAN preservation during IANT. The technique involves wrapping a standard suction catheter around the exposed nerve to facilitate full coverage. This work presents a retrospective review of 31 patients undergoing IANT procedures followed by immediate placement of dental implants between January 2015 and January 2020. IANT was performed either unilaterally or bilaterally on all patients, followed by IAN tubing before implant placement. A total of 46 IANT procedures involving the tubing technique were performed. Overall, 149 dental implants were inserted during IANT surgeries, with a success rate of 98.6%. Sensory disturbance was documented in 47.8% of the treated sites (left/right mandible) at 1 month (22/46 sites), 21.7% at 3 months (10/46 sites), 6.5% at 6 months (3/46 sites), and 2.2% at 12 months (1/46 sites) postimplantation. In total, except for 1 case, sensory disturbance was fully resolved by the end of the 12-month follow-up period. Taken together, the tubing technique described herein is a practical and reproducible method for protecting the IAN during transposition.

Injectable Hydrogels Based on Inter-Polyelectrolyte Interactions between Hyaluronic Acid, Gelatin, and Cationic Cellulose Nanocrystals.

The present work dealt with the development of physically cross-linked injectable hydrogels with potential applications in tissue engineering. The hydrogels were composed of a ternary mixture of a polyanion and a polyampholyte, hyaluronic acid (HA) and gelatin, respectively, bridged by cationic cellulose nanocrystals (cCNCs). A 3D network is formed by employing attractive electrostatic interactions and hydrogen bonding between these components under physiological conditions. The hydrogels demonstrated low viscosity at high stresses, enabling easy injection, structural stability at low stresses (<15 Pa), and nearly complete structure recovery within several minutes. Increasing the cCNC content (>3%) reduced hydrogel swelling and decelerated the degradation in phosphate-buffered saline as compared to that in pure HA and HA-gelatin samples. Biological evaluation of the hydrogel elutions showed excellent cell viability. The proliferation of fibroblasts exposed to elutions of hydrogels with 5% cCNCs reached ∼200% compared to that in the positive control after 11 days. Considering these results, the prepared hydrogels hold great potential in biomedical applications, such as injectable dermal fillers, 3D bioprintable inks, or 3D scaffolds to support and promote soft tissue regeneration.

Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces.

Extracellular vesicles (EVs) have emerged as a promising strategy to promote tissue regeneration. However, overcoming the low EV production yield remains a big challenge in translating EV-based therapies to the clinical practice. Current EV production relies heavily on 2D cell culture, which is not only less physiologically relevant to cells but also requires substantial medium and space. In this study, we engineered tissues seeded with stem cells from dental pulp or adipose tissues, or skeletal muscle cells, and significantly enhanced the EV production yield by applying mechanical stimuli, including flow and stretching, in bioreactors. Further mechanistic investigation revealed that this process was mediated by yes-associated protein (YAP) mechanosensitivity. EVs from mechanically stimulated dental pulp stem cells on 3D scaffolds displayed superior capability in inducing axonal sprouting than the 2D counterparts. Our results demonstrate the promise of this strategy to boost EV production and optimize their functional performance toward clinical translation.

Computer-Assisted Evaluation Confirms Spontaneous Healing of Donor Site One Year following Bone Block Harvesting from Mandibular Retromolar Region-A Cohort Study.

Bone augmentation prior to dental implant placement is a common scenario in the dental implantology field. Among the important intraoral harvesting sites to obtain bone blocks is the ramus/retromolar region that has a high success rate and long-lasting alveolar ridge augmentation. Preserving the bone volume and quality at the donor site is crucial for preventing further complications or to serve as a site for re-harvesting. Healing of the intraoral donor sites has been described in the maxillofacial field. This study aimed to evaluate the spontaneous healing of the mandibular retromolar donor site utilizing computer-assisted quantification 6 and 12 months after bone harvesting. MATERIALS AND METHODS: The study was conducted on patients who underwent an alveolar ridge augmentation using an intraoral retromolar bone graft. Three CBCT scans were performed-intraoperative, and at six months and one year after the surgical procedure. By using the Materialise Mimics Innovation Suite software 26.0 features segmentation by thresholding, Hounsfield unit averaging, and superimposition of the tomographies, we could precisely quantify the healing process utilizing spatial and characteristic measures. RESULTS: In all cases, the computer-aided quantification showed that six months following surgery, the donor site had recovered up to 64.5% ± 4.24 of its initial volume, and this recovery increased to 89.2% ± 2.6 after one year. Moreover, the Hounsfield unit averaging confirmed dynamic bone quality healing, starting at 690.3 ± 81 HU for the bone block, decreasing to 102 ± 27.8 HU at six months postoperatively, and improving to 453.9 ± 91.4 HU at the donor site after a year. CONCLUSIONS: This study demonstrates that there is no need for additional replanting at the donor site following retromolar bone block harvesting, whether autogenous or allograft, since spontaneous healing occurs 12 months following the surgery.

Clinical potential of plasma-functionalized graphene oxide ultrathin sheets for bone and blood vessel regeneration: Insights from cellular and animal models.

Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.

A micro-channel array in a tissue engineered vessel graft guides vascular morphogenesis for anastomosis with self-assembled vascular networks.

Vascularization of 3D engineered tissues poses a great challenge in the field of tissue engineering. One promising approach for vascularizing engineered tissue is cocultivation with endothelial cells (ECs), which spontaneously self-assemble into a natural capillary network in the presence of supportive cells. However, the ECs do not self-assemble according to physiological hierarchy which is required to support blood supply. This work describes the design and fabrication of an AngioTube, a biodegradable engineered macro-vessel surrounded by cylindrical micro-channel array, which is designed to support physiological flow distribution and enable the integration with living capillaries. The well-defined geometry of the engineered micro-channels guides endothelial cells to form patent micro-vessels which sprouted in accordance with the channel orientation. Three different in-vitro models were used to demonstrate anastomosis of these engineered micro-vessels with self-assembled vascular networks. Finally, in-vivo functionality was demonstrated by direct anastomosis with the femoral artery in a rat hindlimb model. This unique approach proposes a new micro-fabrication strategy which introduces uncompromised micro-fluidic device geometrical accuracy at the tissue-scale level. STATEMENT OF SIGNIFICANCE: This study proposes a micro-fabrication strategy suitable for processing real-scale cylindrical implants with very high accuracy, which will enable translation of the high-resolution geometry of micro-fluidic devices to clinically relevant implants containing functional multi-scale vascular networks. Moreover, this approach promises to advance the field of tissue engineering by opening new opportunities to explore the impact of well controlled and uncompromised 3D micro-geometry on cellular behavior.

3D-printable plant protein-enriched scaffolds for cultivated meat development.

Cultivated meat harnesses tissue engineering (TE) concepts to create sustainable, edible muscle tissues, for addressing the rising meat product demands and their global consequences. As 3D-printing is a promising method for creating thick and complex structures, two plant-protein-enriched scaffolding compositions were primarily assessed in our work as 3D-printable platforms for bovine satellite cells (BSC) maturation. Mixtures of pea protein isolate (PPI) and soy protein isolate (SPI) with RGD-modified alginate (Alginate(RGD)) were evaluated as prefabricated mold-based and 3D-printed scaffolds for BSC cultivation, and ultimately, as potential bioinks for cellular printing. Mold-based protein enriched scaffolds exhibited elevated stability and stiffness compared to ones made of Alginate(RGD) alone, while allowing unhindered BSC spreading and maturation. Extrusion based 3D-printing with the two compositions was then developed, while using an edible, removable agar support bath. Successfully fabricated constructs with well-defined geometries supported BSC attachment and differentiation. Finally, cellular bioprinting was demonstrated with PPI-enriched bioinks. Cell recovery post-printing was observed in two cultivation configurations, reaching ∼80-90% viability over time. Moreover, cells could mature within 3D-printed cellular constructs. As animal-derived materials were avoided in our scaffold fabrication process, and pea-protein is known for its low allergic risk, these findings have great promise for further cultivated meat research.

Microcomputed Tomography-Based Analysis of Neovascularization within Bioengineered Vascularized Tissues.

In the field of tissue engineering, evaluating newly formed vascular networks is considered a fundamental step in deciphering the processes underlying tissue development. Several common modalities exist to study vessel network formation and function. However, a proper methodology that allows through three-dimensional visualization of neovessels in a reproducible manner is required. Here, we describe in-depth exploration, visualization, and analysis of vessels within newly formed tissues by utilizing a contrast agent perfusion protocol and high-resolution microcomputed tomography. Bioengineered constructs consisting of porous, biocompatible, and biodegradable scaffolds are loaded with cocultures of adipose-derived microvascular endothelial cells (HAMECs) and dental pulp stem cells (DPSCs) and implanted in a rat femoral bundle model. After 14 days of in vivo maturation, we performed the optimized perfusion protocol to allow host penetrating vascular visualization and assessment within neotissues. Following high-resolution microCT scanning of DPSC:HAMEC explants, we performed the volumetric and spatial analysis of neovasculature. Eventually, the process was repeated with a previously published coculture system for prevascularization based on adipose-derived mesenchymal stromal cells (MSCs) and HAMECs. Overall, our approach allows a comprehensive understanding of vessel organization during engraftment and development of neotissues.

Decline in Maxillofacial Injuries during the Pandemic: The Hidden Face of COVID-19.

Maxillofacial injuries result from a variety of daily activities. Traffic accidents, interpersonal violence, and falls represent some of the most common etiological factors behind maxillofacial fractures. During the COVID-19 outbreak, the social distancing measures imposed by healthcare authorities aimed at abolishing the spread of the viral infection. This study aimed to evaluate the effect of social distancing measures on the incidence of maxillofacial injuries. METHODS: Data were retrieved from the medical file registry at the Galilee Medical Center, Nahariya, Israel. Incidence, gender, age, etiology, and cost of hospitalization during the COVID-19 lockdown and the previous periods were retrieved. RESULTS: A decrease in maxillofacial fractures was registered during the 2020 lockdown; younger patients had the largest share of maxillofacial traumas during this period. The midface was the most involved facial region in both periods, and a reduction of 62.3% in the cost of OMF fracture treatment was observed during the COVID-19 era. CONCLUSIONS: The occurrence, etiology, and cost of treatment of maxillofacial injuries during the COVID-19 period were different from those in the corresponding period in the pre-COVID-19 era. These results can provide a guide to help design programs for the prevention of OMF trauma.

3D Printing and Virtual Surgical Planning in Oral and Maxillofacial Surgery.

Compared to traditional manufacturing methods, additive manufacturing and 3D printing stand out in their ability to rapidly fabricate complex structures and precise geometries. The growing need for products with different designs, purposes and materials led to the development of 3D printing, serving as a driving force for the 4th industrial revolution and digitization of manufacturing. 3D printing has had a global impact on healthcare, with patient-customized implants now replacing generic implantable medical devices. This revolution has had a particularly significant impact on oral and maxillofacial surgery, where surgeons rely on precision medicine in everyday practice. Trauma, orthognathic surgery and total joint replacement therapy represent several examples of treatments improved by 3D technologies. The widespread and rapid implementation of 3D technologies in clinical settings has led to the development of point-of-care treatment facilities with in-house infrastructure, enabling surgical teams to participate in the 3D design and manufacturing of devices. 3D technologies have had a tremendous impact on clinical outcomes and on the way clinicians approach treatment planning. The current review offers our perspective on the implementation of 3D-based technologies in the field of oral and maxillofacial surgery, while indicating major clinical applications. Moreover, the current report outlines the 3D printing point-of-care concept in the field of oral and maxillofacial surgery.

The Modulatory Effect of Adipose-Derived Stem Cells on Endometrial Polyp Fibroblasts.

Endometrial polyps (EPs) are benign overgrowths of the endometrium, with the potential to cause severe complications, ranging from discomfort to inflammation and infertility. Dysfunction of endometrial fibroblasts may be a critical component leading to the development of polyps. Although surgical intervention is the common remedy for severe cases, it comes with drawbacks, including infection, bleeding, and risk of damage to the cervix and adjacent tissues. Adipose-derived mesenchymal stromal cells (ASCs) are at the focus of modern medicine, as key modulators of tissue homeostasis, inflammation, and tissue repair, rendering them prime candidate agents for tissue regeneration and cell-based therapies. In this study, EPs were isolated from patients admitted to the OB/GYN department at the Galilee Medical Center and extracted fibroblasts (endometrial polyp fibroblasts, EPFs) were isolated and characterized. ASCs were isolated from healthy patients. The effect of EPF- and ASC-conditioned media (CM) on polyp-derived fibroblasts was evaluated, in both 2D and 3D assays, as well as on the expression of matrix-related gene expression. Herein, EPFs exposed to ASC-CM exhibited reduced migration, invasion, contraction of hydrogels, and extracellular matrix deposition, compared with those exposed to EPF-CM. Altogether, this study suggests that ASCs may have a modulating effect on fibroblasts involved in forming EPs and may serve as the basis for conservative treatment strategies aimed at treating severe cases of EPs.

Spinal Cord Repair: From Cells and Tissue Engineering to Extracellular Vesicles.

Spinal cord injury (SCI) is a debilitating condition, often leading to severe motor, sensory, or autonomic nervous dysfunction. As the holy grail of regenerative medicine, promoting spinal cord tissue regeneration and functional recovery are the fundamental goals. Yet, effective regeneration of injured spinal cord tissues and promotion of functional recovery remain unmet clinical challenges, largely due to the complex pathophysiology of the condition. The transplantation of various cells, either alone or in combination with three-dimensional matrices, has been intensively investigated in preclinical SCI models and clinical trials, holding translational promise. More recently, a new paradigm shift has emerged from cell therapy towards extracellular vesicles as an exciting "cell-free" therapeutic modality. The current review recapitulates recent advances, challenges, and future perspectives of cell-based spinal cord tissue engineering and regeneration strategies.

3D Bioprinting of Engineered Tissue Flaps with Hierarchical Vessel Networks (VesselNet) for Direct Host-To-Implant Perfusion.

Engineering hierarchical vasculatures is critical for creating implantable functional thick tissues. Current approaches focus on fabricating mesoscale vessels for implantation or hierarchical microvascular in vitro models, but a combined approach is yet to be achieved to create engineered tissue flaps. Here, millimetric vessel-like scaffolds and 3D bioprinted vascularized tissues interconnect, creating fully engineered hierarchical vascular constructs for implantation. Endothelial and support cells spontaneously form microvascular networks in bioprinted tissues using a human collagen bioink. Sacrificial molds are used to create polymeric vessel-like scaffolds and endothelial cells seeded in their lumen form native-like endothelia. Assembling endothelialized scaffolds within vascularizing hydrogels incites the bioprinted vasculature and endothelium to cooperatively create vessels, enabling tissue perfusion through the scaffold lumen. Using a cuffing microsurgery approach, the engineered tissue is directly anastomosed with a rat femoral artery, promoting a rich host vasculature within the implanted tissue. After two weeks in vivo, contrast microcomputer tomography imaging and lectin perfusion of explanted engineered tissues verify the host ingrowth vasculature's functionality. Furthermore, the hierarchical vessel network (VesselNet) supports in vitro functionality of cardiomyocytes. Finally, the proposed approach is expanded to mimic complex structures with native-like millimetric vessels. This work presents a novel strategy aiming to create fully-engineered patient-specific thick tissue flaps.

Human-engineered auricular reconstruction (hEAR) by 3D-printed molding with human-derived auricular and costal chondrocytes and adipose-derived mesenchymal stem cells.

Microtia is a small, malformed external ear, which occurs at an incidence of 1-10 per 10 000 births. Autologous reconstruction using costal cartilage is the most widely accepted surgical microtia repair technique. Yet, the method involves donor-site pain and discomfort and relies on the artistic skill of the surgeon to create an aesthetic ear. This study employed novel tissue engineering techniques to overcome these limitations by developing a clinical-grade, 3D-printed biodegradable auricle scaffold that formed stable, custom-made neocartilage implants. The unique scaffold design combined strategically reinforced areas to maintain the complex topography of the outer ear and micropores to allow cell adhesion for the effective production of stable cartilage. The auricle construct was computed tomography (CT) scan-based composed of a 3D-printed clinical-grade polycaprolactone scaffold loaded with patient-derived chondrocytes produced from either auricular cartilage or costal cartilage biopsies combined with adipose-derived mesenchymal stem cells. Cartilage formation was measured within the constructin vitro, and cartilage maturation and stabilization were observed 12 weeks after its subcutaneous implantation into a murine model. The proposed technology is simple and effective and is expected to improve aesthetic outcomes and reduce patient discomfort.

Prevascularized Scaffolds Bearing Human Dental Pulp Stem Cells for Treating Complete Spinal Cord Injury.

The regeneration of injured spinal cord is hampered by the lack of vascular supply and neurotrophic support. Transplanting tissue-engineered constructs with developed vascular networks and neurotrophic factors, and further understanding the pattern of vessel growth in the remodeled spinal cord tissue are greatly desired. To this end, highly vascularized scaffolds embedded with human dental pulp stem cells (DPSCs) are fabricated, which possess paracrine-mediated angiogenic and neuroregenerative potentials. The potent pro-angiogenic effect of the prevascularized scaffolds is first demonstrated in a rat femoral bundle model, showing robust vessel growth and blood perfusion induced within these scaffolds postimplantation, as evidenced by laser speckle contrast imaging and 3D microCT dual imaging modalities. More importantly, in a rat complete spinal cord transection model, the implantation of these scaffolds to the injured spinal cords can also promote revascularization, as well as axon regeneration, myelin deposition, and sensory recovery. Furthermore, 3D microCT imaging and novel morphometric analysis on the remodeled spinal cord tissue demonstrate substantial regenerated vessels, more significantly in the sensory tract regions, which correlates with behavioral recovery following prevascularization treatment. Taken together, prevascularized DPSC-embedded constructs bear angiogenic and neurotrophic potentials, capable of augmenting and modulating SCI repair.

Rapid prototyping fabrication of soft and oriented polyester scaffolds for axonal guidance.

Biodegradable polyesters have been extensively used for preparation of nerve guidance scaffolds, due to their high biocompatibility and defined degradation periods. However, conventional methods for fabrication of porous polyester scaffolds provide limited control over shape and micro-architecture. Here, a fabrication procedure based on 3D printing was developed to generate highly ordered and anatomically personalized, polyester scaffolds for soft tissue regeneration. Scaffolds composed of Poly-lactic-glycolic acid (PLGA) and poly-L-lactic acid (PLLA) were specifically customized for nerve injuries. This was obtained by using an oriented multi-layer printing pattern which established a linear structure in the fabricated scaffolds to match the aligned topography of nerve tissues. The oriented scaffold was shown to guide regenerating axons to linear conformations and support growth of induced pluripotent stem cell-derived neurons in vitro and in vivo in a model of spinal cord injury. The described scaffolds may advance the field of nerve regeneration. Furthermore, modifications could be integrated to generate soft implants for various types of tissues.

Er:YAG Laser Irradiation Induces Behavioral Changes in V. harveyi.

OBJECTIVE: Laser technologies have many different applications in medical, agricultural, and industrial fields. Studies have shown several effects of laser energy on different bacterial species, in a wide variety of settings. Recent reports have found that one of the unique features of bacteria is their ability to communicate among themselves (quorum sensing). We sought to investigate whether low-energy laser irradiation affects bacterial behavior, which is regulated by quorum sensing. METHODS: Laser irradiations were performed using Er:YAG laser (2940 nm wavelength) at output powers of 0.5, 1.5, 2.5, and 4 W on wild-type Vibrio harveyi. Bioluminescence, motility, and biofilm forming capability were assessed on the bacteria after irradiation. RESULTS: After irradiation of bacteria, positive dose/output power dependencies were found in the bioluminescence omitted from tested experimental groups. Motility of colonies on semi-solid media was inhibited as irradiation output power was increased. However, after irradiation, biomass analysis of biofilm samples showed negligible differences between the irradiated samples and controls. CONCLUSIONS: Results indicate the impact of low-energy laser irradiation on bacterial behavior such as quorum sensing and motility, without affecting bacterial growth patterns.

Comparison of positive-pressure, passive ultrasonic, and laser-activated irrigations on smear-layer removal from the root canal surface.

OBJECTIVE: The purpose of this study was to compare the efficacy of three irrigation techniques for smear-layer removal with 17% EDTA. BACKGROUND DATA: Cleaning and shaping the root canal system during endodontic treatment produces a smear layer and hard tissue debris. Three irrigation techniques were tested for solution infiltration of this layer: positive-pressure irrigation, passive ultrasonic irrigation, and laser-activated irrigation. MATERIALS AND METHODS: Sixty extracted teeth were divided into six equal groups; 17% EDTA was used for 60 sec irrigation of five of the groups. The groups were as follows: Group 1, treated only with ProTaper™ F3 Ni-Ti files; Group 2, positive-pressure irrigation, with a syringe; Group 3, passive ultrasonic irrigation, inserted 1 mm short of the working length; Group 4, passive ultrasonic irrigation, inserted in the upper coronal third of the root; Group 5, Er:YAG laser-activated irrigation, inserted 1 mm short of the working length; and Group 6, Er:YAG laser-activated irrigation, inserted in the upper coronal third of the root. RESULTS: Scanning electron microscopy showed that the smear layer is removed most efficiently using laser-activated irrigation at low energy with 17% EDTA, inserted either at the working length or only in the coronal upper third of the root. Amounts of Ca, P, and O were not significantly different on all treated dentin surfaces. CONCLUSIONS: Smear-layer removal was most effective when the root canals were irrigated using Er:YAG laser at low energy with 17% EDTA solution. Interestingly, removal of the smear layer along the entire canal was similar when the laser was inserted in the upper coronal third and at 1 mm short of the working length of the root canal. This effect was not observed with the ultrasonic and positive-pressure techniques.

Effect of Er:YAG laser-activated irrigation solution on Enterococcus Faecalis biofilm in an ex-vivo root canal model.

UNLABELLED: Abstract Objective: The purpose of this study was to evaluate mineral content and surface morphology of root canals coated with Enterococcus faecalis biofilm after treatment with several endodontic irrigation solutions, with and without Er:YAG laser-activated irrigation (LAI). BACKGROUND DATA: LAI has been introduced as a powerful method for root canal irrigation resulting in smear-layer removal from the root canal wall. METHODS: Distal and palatal roots from 60 freshly extracted human molars were used in this study. The coronal of each tooth was removed. Roots were split longitudinally and placed in an ultrasonic bath to remove the smear layer, creating conditions for the formation of E. faecalis biofilm. After incubation, the two halves were reassembled in impression material to simulate clinical conditions. Specimens were divided into two main groups: roots rinsed with irrigation solutions and roots subjected to laser irradiation combined with irrigation solutions. Solutions tested were 2% chlorhexidine and 17% ethylenediaminetetraacetic acid (EDTA) and saline. RESULTS: Surface morphology: 17% EDTA irrigant solution combined with Er:YAG laser showed the best results for removing bacteria from the root canal walls. Chemical analysis: all samples treated with combined laser irradiation and irrigation solution had low surface levels of Ca compared with samples treated with irrigation alone. The Ca/P ratio was highest in the laser-EDTA group. Overall, mineral changes caused by laser with irrigation solutions were minimal, and statistically nonsignificant. CONCLUSIONS: In vitro irrigation solutions, combined with Er:YAG laser irradiation, were effective in removing E. faecalis biofilm from root canal walls. Irrigation solutions without laser irradiation were less effective, leaving a layer of biofilm on the dentin surface.