Management of an infected immature tooth with a talon cusp using regenerative endodontic treatment: A case report.

OBJECTIVE: Regenerative endodontic treatment (RET) has been considered a successful approach to manage infected immature teeth; however, cases associated with dental anomalies, i.e., talon cusp, need to be further investigated. CASE REPORT: A 7-year-old girl with local swelling associated with the permanent maxillary right central incisor was referred; cone-beam computed tomography revealed a talon cusp, an immature root and two well-defined endodontic lesions. The treatment consisted of regenerative endodontic treatment (RET); the canal was chemically cleaned and a modified triple antibiotic paste was used as intracanal medication. In the next appointment, RET was performed through the creation of blood clot scaffold in the canal covered/sealed with calcium-enriched mixture (CEM) cement. RESULTS: In the 7-day recall session, clinical examination showed that the swelling had completely resolved. At 24-month recall, the treated tooth was asymptomatic and functional. CBCT images demonstrated evidence of maturation in the apical third of the root, healing of two large endodontic lesions and complete dentinal bridge formation beneath CEM cement. CONCLUSION: RET for an infected immature tooth with a dental anomaly, i.e., talon cusp, may be a desirable treatment option and result in the resolution of endodontic lesions as well as regeneration of new vital tissues; allowing continuous root maturation.

The osteogenic differentiation of human dental pulp stem cells in alginate-gelatin/Nano-hydroxyapatite microcapsules.

BACKGROUND: Microcapsule is considered as a promising 3D microenvironment for Bone Tissue Engineering (BTE) applications. Microencapsulation of cells in an appropriate scaffold not only protected the cells against excess stress but also promoted cell proliferation and differentiation. Through the current study, we aimed to microcapsulate the human Dental Pulp Stem Cells (hDPSCs) and evaluated the proliferation and osteogenic differentiation of those cells by using MTT assay, qRT-PCR, Alkaline phosphatase, and Alizarine Red S. RESULTS: The SEM results revealed that Alg/Gel microcapsules containing nHA showed a rough and more compact surface morphology in comparison with the Alg/Gel microcapsules. Moreover, the microencapsulation by Alg/Gel/nHA could improve cell proliferation and induce osteogenic differentiation. The cells cultured in the Alg/Gel and Alg/Gel/nHA microcapsules showed 1.4-fold and 1.7-fold activity of BMP-2 gene expression more in comparison with the control group after 21 days. The mentioned amounts for the BMP-2 gene were 2.5-fold and 4-fold more expression for the Alg/Gel and Alg/Gel/nHA microcapsules after 28 days. The nHA, addition to hDPSCs-laden Alg/Gel microcapsule, could up-regulate the bone-related gene expressions of osteocalcin, osteonectin, and RUNX-2 during the 21 and 28 days through the culturing period, too. Calcium deposition and ALP activities of the cells were observed in accordance with the proliferation results as well as the gene expression analysis. CONCLUSION: The present study demonstrated that microencapsulation of the hDPSCs inside the Alg/Gel/nHA hydrogel could be a potential approach for regenerative dentistry in the near future.

Chemical binding of pyrrolidinyl peptide nucleic acid (acpcPNA-T9) probe with AuNPs toward label-free monitoring of miRNA-21: A novel biosensing platform for biomedical analysis and POC diagnostics.

miRNAs are attractive factors in cancer research studies due to their important roles for regulating of gene expression. Because of miRNA-21 expression surplus in many types of cancers, so accurate identification is important. Increasing efforts have caused different methods to improve the sensitivity and specificity of detection. Present study is an attempt to report a new electrochemical label-free PNA-based bioassay for detection of miRNA-21. In this study, gold electrode was modified by gold nanoparticles to improve a functional PNA-based biosensor. The EDS and field emission scanning electron microscope (FE-SEM) were used to detect fabrication of biosensor. The electrochemical behavior of sensor was evaluated after inserting of acpcPNA probes and miRNA-21 on the stucture of electrode and analyzed essential parameters such as various concentration of target miRNA, hybridization time, reproducibility, stability, and applicability. The results of study demonstrated that engineered biosensor was successfully fabricated. The findings showed the highest amount of current in 5 minutes hybridization time, with suitable reproducibility and stability. This innovative miRNA-based biosensor presents a sensitive and specific method in fast and may be lab-on chip assay in future.

Electrochemical immunoplatform to assist in the diagnosis of oral cancer through the determination of CYFRA 21.1 biomarker in human saliva samples: Preparation of a novel portable biosensor toward non-invasive diagnosis of oral cancer.

In this study, a novel, low-cost, and flexible paper-based electrochemical immunosensor was developed for the bioanalysis of Cyfra 21.1 biomarker in human saliva samples by using stabilization of synthesis Ag nano-ink on the surface of paper using pen-on-paper technology. The employed electrochemical techniques for the evaluation of immunoplatform performance were differential pulse voltammetry and chronoamperometry. Also, the prepared immunosensor showed great ability in the determination of Cyfra21.1 in human saliva specimens. Under the optimized conditions, the obtained linear range was from 0.0025 to 10 ng/mL, and the obtained LLOQ was 0.0025 ng/mL. The developed immunosensor is easy to prepare, sensitive, cost-effective, portable, and simple. So proposed immunoplatform can be an accomplished biodevice in clinical laboratories. The proposed paper-based immunosensor could be a hopefully new and cheap tool for the diagnosis of other biomarkers. Also, the prepared immunosensor showed great ability in the determination of Cyfra21.1 biomarker in human saliva specimens.

Bioactive polymeric scaffolds for osteogenic repair and bone regenerative medicine.

The loss of bone tissue is a striking challenge in orthopedic surgery. Tissue engineering using various advanced biofunctional materials is considered a promising approach for the regeneration and substitution of impaired bone tissues. Recently, polymeric supportive scaffolds and biomaterials have been used to rationally promote the generation of new bone tissues. To restore the bone tissue in this context, biofunctional polymeric materials with significant mechanical robustness together with embedded materials can act as a supportive matrix for cellular proliferation, adhesion, and osteogenic differentiation. The osteogenic regeneration to replace defective tissues demands greater calcium deposits, high alkaline phosphatase activity, and profound upregulation of osteocalcin as a late osteogenic marker. Ideally, the bioactive polymeric scaffolds (BPSs) utilized for bone tissue engineering should impose no detrimental impacts and function as a carrier for the controlled delivery and release of the loaded molecules necessary for the bone tissue regeneration. In this review, we provide comprehensive insights into different synthetic and natural polymers used for the regeneration of bone tissue and discuss various technologies applied for the engineering of BPSs and their physicomechanical properties and biological effects.

The effect of electromagnetic fields on survival and proliferation rate of dental pulp stem cells.

Aims: Extremely low-frequency electromagnetic fields (ELF-EMF) can affect biological systems and alter some cell functions like proliferation rate. Dental pulp tissue is known as a source of multipotent stromal stem cells (MSCs), which can be obtained by a less invasive and more available process compared to bone marrow-derived stem cells (BMSCs). This study aimed to consider the effect of ELF-EMF on proliferation rates of human dental pulp stem cells (hDPSCs).Material and methods: ELF-EMF was generated by a system including autotransformer, multi-meter, solenoid coils, teslameter and its probe. The effect of ELF-EMF with the intensity of 0.5 and 1 mT and 50 Hz on the proliferation rate of hDPSCs was assessed in 20 and 40 min per day for 7 days. MTT assay and DAPI test were used to determine the growth and proliferation of DPSCs.Results: Based on MTT, ELF-EMF has maximum effect with the intensity of 1 mT for 20 min/day on the proliferation of hDPSCs. The survival and proliferation rate in all exposure groups were significantly higher than the control group. Based on the data obtained from MTT and DAPI assay, the number of viable cells in the group exposed to 1 mT for 20 min/day was higher than other groups (p < .05).Conclusions: Regarding to the results of this study, 0.5 and 1 mT ELF-EMF can enhance survival and proliferation rates of hDPSCs.

The Use of Nanomaterials in Tissue Engineering for Cartilage Regeneration; Current Approaches and Future Perspectives.

The repair and regeneration of articular cartilage represent important challenges for orthopedic investigators and surgeons worldwide due to its avascular, aneural structure, cellular arrangement, and dense extracellular structure. Although abundant efforts have been paid to provide tissue-engineered grafts, the use of therapeutically cell-based options for repairing cartilage remains unsolved in the clinic. Merging a clinical perspective with recent progress in nanotechnology can be helpful for developing efficient cartilage replacements. Nanomaterials, < 100 nm structural elements, can control different properties of materials by collecting them at nanometric sizes. The integration of nanomaterials holds promise in developing scaffolds that better simulate the extracellular matrix (ECM) environment of cartilage to enhance the interaction of scaffold with the cells and improve the functionality of the engineered-tissue construct. This technology not only can be used for the healing of focal defects but can also be used for extensive osteoarthritic degenerative alterations in the joint. In this review paper, we will emphasize the recent investigations of articular cartilage repair/regeneration via biomaterials. Also, the application of novel technologies and materials is discussed.

Efficacy of mental-incisive nerve block in root canal treatment of mandibular first molars with asymptomatic irreversible pulpitis: a randomized controlled trial.

The aim of this study was to evaluate the effect of mental-incisive nerve block (MINB) along with finger pressure following inferior alveolar nerve block (IANB) on anesthetic success in mandibular first molars with asymptomatic irreversible pulpitis. In this randomized controlled trial, 70 patients were randomly divided into 2 groups (n = 35). Each patient in the control group received only a standard IANB injection of 1.8 mL of 2% lidocaine with 1:100,000 epinephrine. The injection was administered within 1 minute, using a standard aspirating dental cartridge fitted with a 27-gauge dental needle. In the intervention group, 15 minutes after injection of the standard IANB as described for the control group, each patient received a standard MINB injection of 1 mL of 2% lidocaine containing 1:100,000 epinephrine, administered by an operator not involved in assessing the outcomes. After the MINB injection, the patient applied firm finger pressure to the soft tissue of the mental foramen region for 1 minute using the hand on the side opposite to the injection. Objective assessment of tooth anesthesia was carried out with electric pulp tests (EPTs). In addition, the patients rated their pain during the initial steps of endodontic treatment based on a visual analog scale (VAS). The Mann-Whitney U and Wilcoxon tests were used for the analysis of data. Of the 35 patients in each group, 20.0% (7 patients) in the control group and 71.4% (25 patients) in the intervention group had no response to EPTs 15 minutes after injections; this difference was statistically significant (P < 0.05). The VAS pain scores were significantly higher in the control group than in the intervention group (P = 0.001). The administration of MINB with pressure following IANB significantly improved the success of anesthesia in mandibular first molars with asymptomatic irreversible pulpitis.

Retraction Note to: Synthesis, characterization and in vitro studies of doxorubicin-loaded magnetic nanoparticles grafted to smart copolymers on A549 lung cancer cell line.

The Editors have retracted this article [1] because Figs. 6a and 6c have been used in three other publications to represent scanning electron micrographs of different nanoparticles [2-4]. The data reported in this article are therefore unreliable. In addition, Fig. 3 was reproduced from [5] with retrospective permission and the credit line should read as follows: "Reprinted from Acta Biomaterialia, Volume 3, Zhang, J. and Misra, R.D.K., Magnetic drug-targeting carrier encapsulated with thermosensitive smart polymer: core-shell nanoparticle carrier and drug release response, pp. 838-850, copyright (2007) with permission from Acta Materialia Inc. Authors Abolfazl Akbarzadeh, Maryam Anzaby, Soodabeh Davaran, Sang Woo Joo and Mohammad Samiei agree to this retraction. Authors Younes Hanifehpour and Hamid Tayefi Nasrabadi have not responded to any correspondence about this retraction.

Stem Cell Therapy: Curcumin Does the Trick.

Curcumin is a dietary polyphenol and a bioactive phytochemical agent that possesses anti-inflammatory, antioxidant, anticancer, and chemopreventive properties. Some of the predominant activities of stem cells include regeneration of identical cells and the ability to maintain the proliferation and multipotentiality. However, these cells could be stimulated to differentiate into specific cell types. Curcumin protects some stem cells from toxicity and can stimulate proliferation and differentiation of stem cells. In the present review, we summarize the antioxidant, stemness activity, antiaging, and neuroprotective as well as wound healing and regenerative effects of curcumin.

Silver nanoparticles: Synthesis methods, bio-applications and properties.

Silver nanoparticles size makes wide range of new applications in various fields of industry. Synthesis of noble metal nanoparticles for applications such as catalysis, electronics, optics, environmental and biotechnology is an area of constant interest. Two main methods for Silver nanoparticles are the physical and chemical methods. The problem with these methods is absorption of toxic substances onto them. Green synthesis approaches overcome this limitation. Silver nanoparticles size makes wide range of new applications in various fields of industry. This article summarizes exclusively scalable techniques and focuses on strengths, respectively, limitations with respect to the biomedical applicability and regulatory requirements concerning silver nanoparticles.

Does prenatal restraint stress change the craniofacial growth pattern of rat offspring?

A major and frequently encountered condition underlying the long-term programming effects of the intrauterine environment is exposure to stress. Gestational stress is an environmental factor that induces physical and behavioral alterations in offspring. Seventy female virgin Wistar rats were mated with one male rat for a maximum of four times, after which 52 pregnant rats were divided into two groups. In the experimental group the rats were exposed to restraint stress during pregnancy, whereas the control group did not receive the stress protocol. One male litter was randomly chosen from the offspring of each rat with 8-13 pups. A total of 40 male rat offspring were available for analysis. Thirty-one linear and angular measurements were analyzed in both study groups to investigate whether prenatal restraint stress changes the craniofacial growth pattern of rat offspring. In the prenatally stressed group, anterior cranial base length and viscerocranium measures were significantly increased compared with the control group, whereas cranial width, mandibular dimensions, and posterior cranial height and length remained unchanged. Furthermore, the prenatally stressed group showed backward rotation of the midface and decreased flattening of the cranial vault. It was concluded that prenatal chronic stress can induce alterations in the craniofacial growth pattern by promoting endochondral growth in the cranial base and nasal septum.

A randomized trial of direct pulp capping in primary molars using MTA compared to 3Mixtatin: a novel pulp capping biomaterial.

OBJECTIVE: This study aimed to examine the efficacy of 3Mixtatin (a combination of simvastatin and 3Mix antibiotic) as a novel pulp capping biomaterial in DPC of human primary molars. METHODS: In this randomized clinical trial, 160 primary molars from 83 healthy children aged 3-6 years were randomly allocated into four groups. Small traumatic non-caries pulpal exposures were treated by DPC using simvastatin, 3Mix, 3Mixtatin, or MTA. Capping materials were covered with hard-setting zinc oxide eugenol (ZOE) cement, and then, teeth were restored with amalgam. Clinical and radiographic examinations were conducted at 2, 6, and 12 months after treatment. The data were compared using chi-square test at a significance level of 0.05. RESULTS: One hundred and twenty-nine teeth were available for follow-up study. By the end of 12 months, the overall success rates were 93.8% in MTA, 91.9% in 3Mixtatin, 62.5% in 3Mix, and 57.1% in simvastatingroups. No statistically significant difference was found between the outcomes of MTA and 3Mixtatin groups (P > 0.05). 3Mixtatin had statistically superior results compared to 3Mix and simvastatin (P < 0.01). CONCLUSION: Radiographic and clinical outcomes in 3Mixtatin group could suggest it as an acceptable alternative in DPC of primary molar teeth.

Impact of Chlorhexidine Pretreatment Followed by Probiotic Streptococcus salivarius Strain K12 on Halitosis in Children: A Randomised Controlled Clinical Trial.

PURPOSE: The aim of this study was to examine the effect of chlorhexidine disinfection, as a chemical method of oral hygiene practice, and subsequent use of probiotics on halitosis in children. The effects of mechanical and chemical oral hygiene practice methods on the severity of halitosis were also assessed. MATERIALS AND METHODS: 208 children with organoleptic test (OLT) scores of 2 or more were randomly assigned to four groups: A: conventional oral hygiene practices (COH) including toothbrushing and flossing; B: COH + tongue scraping (TS); C: COH + TS + chlorhexidine; D: COH + TS + chlorhexidine + probiotics. OLT was performed at 1-week and 3-month follow-ups. RESULTS: A significant and stable number of participants showed major and moderate levels of improvement in OLT scores in group D (p < 0.001). The improvement of OLT scores in group C was also significant (p < 0.001), but not stable over the follow-ups (p = 0.44). Neither significant nor stable improvements in the OLT scores were detected in groups A and B through follow-ups (p > 0.05). CONCLUSION: Probiotic therapy following oral disinfection with chlorhexidine may reduce the severity of halitosis over longer periods.

Scanning electron microscopy comparison of the cleaning efficacy of a root canal system by Nd:YAG laser and rotary instruments.

This study evaluated the cleaning efficacy of a root canal system by Nd:YAG laser and rotary instruments. Sixty single-rooted human teeth were divided into four experimental groups (n=15). In the first group the teeth were prepared with a step-back technique using conventional K-files. In the second and third groups, tooth preparation was carried out using Nd:YAG laser and rotary NiTi instruments, respectively. Teeth in the fourth group were prepared by combined laser and rotary methods. The smear layer remaining on canal walls was then assessed by scanning electron microscopy in the coronal, middle, and apical portions. The comparison of smear layer removal efficacy between groups was carried out by Kruskal-Wallis and Mann-Whitney U tests. The mean grades of smear layer removal in rotary-laser, rotary, laser and step-back techniques were 1.34 ± 0.18, 2.2 ± 0.28, 1.91 ± 0.25, and 2.42 ± 0.19, respectively. On the whole, differences between rotary-laser and rotary groups, step-back, and the three other techniques (rotary, laser, and rotary-laser) were significant at p=0.034. Based on the findings of this study, the cleaning efficacy of rotary, laser, and rotary-laser techniques were better than the step-back technique and the combined laser and rotary technique was the most efficient method.

Ultrashort hybrid metal-insulator plasmonic directional coupler.

An ultrashort plasmonic directional coupler based on the hybrid metal-insulator slab waveguide is proposed and analyzed at the telecommunication wavelength of 1550 nm. It is first analyzed using the supermode theory based on mode analysis via the transfer matrix method in the interaction region. Then the 2D model of the coupler, including transition arms, is analyzed using a commercial finite-element method simulator. The hybrid slab waveguide is composed of a metallic layer of silver and two dielectric layers of silica (SiO2) and silicon (Si). The coupler is optimized to have a minimum coupling length and to transfer maximum power considering the layer thicknesses as optimization variables. The resulting coupling length in the submicrometer region along with a noticeable power transfer efficiency are advantages of the proposed coupler compared to previously reported plasmonic couplers.

Effect of Silver Nanoparticles of Herbal Origin on the Compressive and Push-out Bond Strengths of Mineral Trioxide Aggregate.

Introduction: The purpose of this in vitro study was to investigate the effect of incorporating silver nanoparticles (AgNPs) of herbal origin into mineral trioxide aggregate (MTA) on the push-out bond strength (PBS) and compressive strength (CS) in simulated furcal area perforations. Materials and Methods: In this in vitro study, simulated furcal area perforations (1.3 mm in diameter and 2 mm in depth) were created in 40 extracted human lower molar teeth, which were divided into two groups (n=20): MTA alone and MTA combined with AgNPs (2% wt). Using a universal testing machine, PBS was evaluated by performing push-out tests, while CS was assessed using cylindrical specimens. The normal distribution of data was checked using the Kolmogorov-Smirnov test, and statistical analysis was performed using two-way ANOVA. Results: The CS results showed no significant difference between the MTA group at 4 and 21 days (P=0.297), but a significant difference was observed in the nanosilver/MTA group (P=0.013). However, there was no significant difference in the push-out bond strength among the study groups (P>0.05). Conclusion: The incorporation of herbal origin silver nanoparticles did not significantly affect the PBS or CS of MTA.

Effect of Incorporating Titanium Dioxide Nanoparticles into White Portland Cement, White Mineral Trioxide Aggregate, and Calcium Enriched Mixture Cement on the Push-out Bond Strength to Furcal Area Dentin.

Statement of the Problem: Bond strength of furcation repair materials is an essential factor in clinical success. Studies on the effect of adding titanium dioxide (TiO2) nanoparticles on the push-out bond strength of commonly used endodontic cements for furcation perforation repair is limited. Purpose: This study aimed to evaluate the effect of adding TiO2 nanoparticles to white Portland cement (PC), white mineral trioxide aggregate (MTA), and calcium enriched mixture cement (CEM) on their push-out bond strengths. Materials and Method: In this in vitro study, 120 endodontically treated molars were assigned to six groups (n=20) based on the material used to repair the perforation. In three groups, the cements (white PC, white MTA, and CEM) were placed in pure form, and in the three remaining groups, 1 weight % of TiO2 was added. The push-out bond strength was measured using a universal testing machine at a strain rate of 0.5 mm/min. Data were analyzed using one-way ANOVA and post hoc Games-Howell test (p< 0.05). Results: One-way ANOVA showed significant differences in the mean bond strength values between the six groups (p= 0.002). The post hoc Games-Howell test showed that the bond strengths in MTA+TiO2 and PC+TiO2 groups were significantly higher than those in MTA and PC groups, respectively. However, there was no significant difference in the bond strength between CEM and CEM+ TiO2 groups. Conclusion: The incorporation of TiO2 into MTA and PC increased their push-out bond strength. However, it did not affect the push-out bond strength of CEM cement.

Synthesis, characterization, and evaluation of Hesperetin nanocrystals for regenerative dentistry.

Hesperetin (HS), a metabolite of hesperidin, is a polyphenolic component of citrus fruits. This ingredient has a potential role in bone strength and the osteogenic differentiation. The bone loss in the orofacial region may occur due to the inflammation response of host tissues. Nanotechnology applications have been harshly entered the field of regenerative medicine to improve the efficacy of the materials and substances. In the current study, the hesperetin nanocrystals were synthesized and characterized. Then, the anti-inflammatory and antioxidative effects of these nanocrystals were evaluated on inflamed human Dental Pulp Stem Cells (hDPSCs) and monocytes (U937). Moreover, the osteoinduction capacity of these nanocrystals was assessed by gene and protein expression levels of osteogenic specific markers including RUNX2, ALP, OCN, Col1a1, and BSP in hDPSCs. The deposition of calcium nodules in the presence of hesperetin and hesperetin nanocrystals was also assessed. The results revealed the successful fabrication of hesperetin nanocrystals with an average size of 100 nm. The levels of TNF, IL6, and reactive oxygen species (ROS) in inflamed hDPSCs and U937 significantly decreased in the presence of hesperetin nanocrystals. Furthermore, these nanocrystals induced osteogenic differentiation in hDPSCs. These results demonstrated the positive and effective role of fabricated nanocrystal forms of this natural ingredient for regenerative medicine purposes.

Injectable thermosensitive chitosan/gelatin hydrogel for dental pulp stem cells proliferation and differentiation.

Introduction: Biocompatible and biodegradable scaffolds based on natural polymers such as gelatin and chitosan (CS) provide suitable microenvironments in dental tissue engineering. In the present study, we report on the synthesis of injectable thermosensitive hydrogel (PNIPAAm-g-CS copolymer/gelatin hybrid hydrogel) for osteogenic differentiation of human dental pulp stem cells (hDPSCs). Methods: The CS-g-PNIPAAm was synthesized using the reaction of carboxyl terminated PNIPAAm with CS, which was then mixed with various amounts of gelatin solution in the presence of genipin as a chemical crosslinker to gain a homogenous solution. The chemical composition and microstructures of the fabricated hydrogels were confirmed by FT-IR and SEM analysis, respectively. To evaluate the mechanical properties (e.g., storage and loss modulus of the gels), the rheological analysis was considered. Calcium deposition and ALP activity of DPSCs were carried out using alizarin red staining and ALP test. While the live/dead assay was performed to study its toxicity, the real-time PCR was conducted to investigate the osteogenic differentiation of hDPSCs cultured on prepared hydrogels. Results: The hydrogels with higher gelatin incorporation showed a slightly looser network compared to the other ones. The hydrogel with less gelatin indicates a rather higher value of G', indicating a higher elasticity due to more crosslinking reaction of amine groups of CS via a covalent bond with genipin. All the hydrogels contained viable cells with negligible dead cells, indicating the high biocompatibility of the prepared hydrogels for hDPSCs. The quantitative results of alizarin red staining displayed a significant rise in calcium deposition in hDPSCs cultured on prepared hydrogels after 21 days. Further, hDPSCs cultured on hydrogel with more gelatin displayed the most ALP activity. The expression of late osteogenic genes such as OCN and BMP-2 were respectively 6 and 4 times higher on the hydrogel with more gelatin than the control group after 21 days. Conclusion: The prepared PNIPAAm-g-CS copolymer/gelatin hybrid hydrogel presented great features (e.g., porous structure, suitable rheological behavior, and improved cell viability), and resulted in osteogenic differentiation necessary for dental tissue engineering.