Craniofacial Tissue Regeneration Research Articles

Effects of Innervation on Angiogenesis and Osteogenesis in Bone and Dental Tissue Engineering.

The repair and regeneration of critical-sized bone defects remain an urgent challenge. Bone tissue engineering represents an exciting solution for regeneration of large bone defects. Recently, the importance of innervation in tissue-engineered bone regeneration has been increasingly recognized. The cross talk between nerve and bone provides important clues for bone repair and regeneration. Furthermore, the promotion of angiogenesis by innervation can accelerate new bone formation. However, the mechanisms involved in the promotion of vascular and bone regeneration by the nervous system have not yet been established. In addition, simultaneous neurogenesis and vascularization in bone tissue engineering have not been fully investigated. This article represents the first review on the effects of innervation in enhancing angiogenesis and osteogenesis in bone and dental tissue engineering. Cutting-edge research on the effects of innervation through biomaterials on bone and dental tissue repairs is reviewed. The effects of various nerve-related factors and cells on bone regeneration are discussed. Finally, novel clinical applications of innervation for bone, dental, and craniofacial tissue regeneration are also examined.

Cellular Senescence in Craniofacial Tissue Regeneration: Inducers, Biomarkers, and Interventions.

Craniofacial defects and dental tissue loss have significant negative impacts on the structure and function of jaws and face, often resulting in psychological issues in patients, emphasizing the urgent need for effective craniofacial tissue reconstruction. Unfortunately, natural regeneration of these tissues is limited. Dental-derived mesenchymal stem cells (MSCs) have emerged as a promising resource for tissue engineering-based therapeutic approaches. However, the clinical outcomes of MSC-based transplantation have not met expectations due to various complex reasons, and cellular senescence is recognized as one of the potential mechanisms contributing to the suboptimal results. The quality of MSC decreases during large-scale in vitro expansion, and it is also influenced by the age and the health status of donors. To address these challenges, extensive efforts have been made to developing strategies to combat senescence in tissue engineering, leveraging on current knowledge of underlying mechanisms. This review aims to elucidate the impact of cell senescence in craniofacial and dental regeneration and provides an overview of state-of-the-art antisenescence strategies. We first discuss the potential factors that trigger cell senescence in craniofacial tissue engineering. Then we describe senescence biomarkers, monitoring methods for senescent MSCs, and their underlying molecular mechanisms. The primary focus of this review is on current strategies to inhibit and alleviate cell senescence in tissue engineering. We summarize the strategies concerning the prevention of cell senescence, senolysis, modulation of the senescent associated secretory phenotype, and reversal of senescent MSCs, offering promising opportunities to overcome the challenges associated with cell senescence in craniofacial tissue engineering.

Exploring the Use of Animal Models in Craniofacial Regenerative Medicine: A Narrative Review.

The craniofacial region contains skin, bones, cartilage, the temporomandibular joint (TMJ), teeth, periodontal tissues, mucosa, salivary glands, muscles, nerves, and blood vessels. Applying tissue engineering therapeutically helps replace lost tissues after trauma or cancer. Despite recent advances, it remains essential to standardize and validate the most appropriate animal models to effectively translate preclinical data to clinical situations. Therefore, this review focused on applying various animal models in craniofacial tissue engineering and regeneration. This research was based on PubMed, Scopus, and Google Scholar data available until January 2023. This study included only English-language publications describing animal models' application in craniofacial tissue engineering (in vivo and review studies). Study selection was based on evaluating titles, abstracts, and full texts. The total number of initial studies was 6454. Following the screening process, 295 articles remained on the final list. Numerous in vivo studies have shown that small and large animal models can benefit clinical conditions by assessing the efficacy and safety of new therapeutic interventions, devices, and biomaterials in animals with similar diseases/defects to humans. Different species' anatomical, physiologic, and biological features must be considered in developing innovative, reproducible, and discriminative experimental models to select an appropriate animal model for a specific tissue defect. As a result, understanding the parallels between human and veterinary medicine can benefit both fields.

Mesenchymal Stem Cells in Soft Tissue Regenerative Medicine: A Comprehensive Review.

Soft tissue regeneration holds significant promise for addressing various clinical challenges, ranging from craniofacial and oral tissue defects to blood vessels, muscle, and fibrous tissue regeneration. Mesenchymal stem cells (MSCs) have emerged as a promising tool in regenerative medicine due to their unique characteristics and potential to differentiate into multiple cell lineages. This comprehensive review explores the role of MSCs in different aspects of soft tissue regeneration, including their application in craniofacial and oral soft tissue regeneration, nerve regeneration, blood vessel regeneration, muscle regeneration, and fibrous tissue regeneration. By examining the latest research findings and clinical advancements, this article aims to provide insights into the current state of MSC-based therapies in soft tissue regenerative medicine.

Applications of Biotechnology to the Craniofacial Complex: A Critical Review.

Biotechnology shows a promising future in bridging the gap between biomedical basic sciences and clinical craniofacial practice. The purpose of the present review is to investigate the applications of biotechnology in the craniofacial complex. This critical review was conducted by using the following keywords in the search strategy: "biotechnology", "bioengineering", "craniofacial", "stem cells", "scaffolds", "biomarkers", and "tissue regeneration". The databases used for the electronic search were the Cochrane Library, Medline (PubMed), and Scopus. The search was conducted for studies published before June 2022. The applications of biotechnology are numerous and provide clinicians with the great benefit of understanding the etiology of dentofacial deformities, as well as treating the defected areas. Research has been focused on craniofacial tissue regeneration with the use of stem cells and scaffolds, as well as in bioinformatics with the investigation of growth factors and biomarkers capable of providing evidence for craniofacial growth and development. This review presents the biotechnological opportunities in the fields related to the craniofacial complex and attempts to answer a series of questions that may be of interest to the reader. Biotechnology seems to offer a bright future ahead, improving and modernizing the clinical management of cranio-dento-facial diseases. Extensive research is needed as human studies on this subject are few and have controversial results.

Small molecule-mediated regenerative engineering for craniofacial and dentoalveolar bone.

The comprehensive reconstruction of extensive craniofacial and dentoalveolar defects remains a major clinical challenge to this day, especially in complex medical cases involving cancer, cranioplasty, and traumatic injury. Currently, osteogenic small molecule-based compounds have been explored extensively to repair and regenerate bone tissue because of their unique advantages. Over the past few years, a number of small molecules with the potential of craniofacial and periodontal bone tissue regeneration have been reported in literature. In this review, we discuss current progress using small molecules to regulate cranial and periodontal bone regeneration. Future directions of craniofacial bone regenerative engineering using the small molecule-based compounds will be discussed as well.

Biomedical applications of three-dimensional bioprinted craniofacial tissue engineering.

Anatomical complications of the craniofacial regions often present considerable challenges to the surgical repair or replacement of the damaged tissues. Surgical repair has its own set of limitations, including scarcity of the donor tissues, immune rejection, use of immune suppressors followed by the surgery, and restriction in restoring the natural aesthetic appeal. Rapid advancement in the field of biomaterials, cell biology, and engineering has helped scientists to create cellularized skeletal muscle-like structures. However, the existing method still has limitations in building large, highly vascular tissue with clinical application. With the advance in the three-dimensional (3D) bioprinting technique, scientists and clinicians now can produce the functional implants of skeletal muscles and bones that are more patient-specific with the perfect match to the architecture of their craniofacial defects. Craniofacial tissue regeneration using 3D bioprinting can manage and eliminate the restrictions of the surgical transplant from the donor site. The concept of creating the new functional tissue, exactly mimicking the anatomical and physiological function of the damaged tissue, looks highly attractive. This is crucial to reduce the donor site morbidity and retain the esthetics. 3D bioprinting can integrate all three essential components of tissue engineering, that is, rehabilitation, reconstruction, and regeneration of the lost craniofacial tissues. Such integration essentially helps to develop the patient-specific treatment plans and damage site-driven creation of the functional implants for the craniofacial defects. This article is the bird's eye view on the latest development and application of 3D bioprinting in the regeneration of the skeletal muscle tissues and their application in restoring the functional abilities of the damaged craniofacial tissue. We also discussed current challenges in craniofacial bone vascularization and gave our view on the future direction, including establishing the interactions between tissue-engineered skeletal muscle and the peripheral nervous system.

Skeletal Stem Cell Isolation from Cranial Suture Mesenchyme and Maintenance of Stemness in Culture.

Skeletal stem cells residing in the suture mesenchyme are responsible for calvarial development, homeostatic maintenance, and injury-induced repair. These naïve cells exhibit long-term self-renewal, clonal expansion, and multipotency. They possess osteogenic abilities to regenerate bones in a cell-autonomous manner and can directly replace the damaged skeleton. Therefore, the establishment of reliable isolation and culturing methods for skeletal stem cells capable of preserving their stemness promises to further explore their use in cell-based therapy. Our research team is the first to isolate and purify skeletal stem cells from the calvarial suture and demonstrate their potent ability to generate bone at a single-cell level. Here, we describe detailed protocols for suture stem cell (SuSC) isolation and stemness maintenance in culture. These methods are extremely valuable for advancing our knowledge base of skeletal stem cells in craniofacial development, congenital deformity, and tissue repair and regeneration.

Rabbit calvarial and mandibular critical-sized bone defects as an experimental model for the evaluation of craniofacial bone tissue regeneration

IntroductionMany studies have aimed to investigate the regeneration potential of bone substitutes through animal models at different defect sites, where the bone healing mechanism varies due to developmental, structural and functional differences. This study aims to develop a rabbit model with two functionally different (non-load-bearing calvarias and load-bearing mandibular) critical-sized defects (CSD) in one rabbit. Material & MethodThe comparison of the “gold standard” autograft to a sham (no graft) control was undertaken in order to validate this model; at the same time, a 3D-printed biphasic calcium phosphate scaffold was implanted to test its utility in the evaluation of new bone substitute materials. Twenty rabbits were selected with both a 10 mm calvaria defect and a 11 mm bicortical semi-cylindrical mandibular defect. The animals were euthanized at 4 and 12 weeks once surgery, microcomputed tomography and histological analysis had been performed. ResultsIn the case of the calvaria, the results for the non-healing sham group compared with the healing of those that had undergone the autograft validated the CSD model. But the mandibular defect was not validated, due to the particularity of mandible high mechanical stress and infectious risk. DiscussionThis study showed for the first time that rabbits have a high tolerance for the bilateral double-site CSD model under consideration; and further studies are essential to modify and improve the design of mandibular CSD.

Tissue Engineered Neurovascularization Strategies for Craniofacial Tissue Regeneration.

Craniofacial tissue injuries, diseases, and defects, including those within bone, dental, and periodontal tissues and salivary glands, impact an estimated 1 billion patients globally. Craniofacial tissue dysfunction significantly reduces quality of life, and successful repair of damaged tissues remains a significant challenge. Blood vessels and nerves are colocalized within craniofacial tissues and act synergistically during tissue regeneration. Therefore, the success of craniofacial regenerative approaches is predicated on successful recruitment, regeneration, or integration of both vascularization and innervation. Tissue engineering strategies have been widely used to encourage vascularization and, more recently, to improve innervation through host tissue recruitment or prevascularization/innervation of engineered tissues. However, current scaffold designs and cell or growth factor delivery approaches often fail to synergistically coordinate both vascularization and innervation to orchestrate successful tissue regeneration. Additionally, tissue engineering approaches are typically investigated separately for vascularization and innervation. Since both tissues act in concert to improve craniofacial tissue regeneration outcomes, a revised approach for development of engineered materials is required. This review aims to provide an overview of neurovascularization in craniofacial tissues and strategies to target either process thus far. Finally, key design principles are described for engineering approaches that will support both vascularization and innervation for successful craniofacial tissue regeneration.

Potential of Bone-Marrow-Derived Mesenchymal Stem Cells for Maxillofacial and Periodontal Regeneration: A Narrative Review.

Bone-marrow-derived mesenchymal stem cells (BM-MSCs) are one of the most widely studied postnatal stem cell populations and are considered to utilize more frequently in cell-based therapy and cancer. These types of stem cells can undergo multilineage differentiation including blood cells, cardiac cells, and osteogenic cells differentiation, thus providing an alternative source of mesenchymal stem cells (MSCs) for tissue engineering and personalized medicine. Despite the ability to reprogram human adult somatic cells to induced pluripotent stem cells (iPSCs) in culture which provided a great opportunity and opened the new door for establishing the in vitro disease modeling and generating an unlimited source for cell base therapy, using MSCs for regeneration purposes still have a great chance to cure diseases. In this review, we discuss the important issues in MSCs biology including the origin and functions of MSCs and their application for craniofacial and periodontal tissue regeneration, discuss the potential and clinical applications of this type of stem cells in differentiation to maxillofacial bone and cartilage in vitro, and address important future hopes and challenges in this field.

Tracking of Oral and Craniofacial Stem Cells in Tissue Development, Regeneration, and Diseases.

The craniofacial region hosts a variety of stem cells, all isolated from different sources of bone and cartilage. However, despite scientific advancements, their role in tissue development and regeneration is not entirely understood. The goal of this review is to discuss recent advances in stem cell tracking methods and how these can be advantageously used to understand oro-facial tissue development and regeneration. Stem cell tracking methods have gained importance in recent times, mainly with the introduction of several molecular imaging techniques, like optical imaging, computed tomography, magnetic resonance imaging, and ultrasound. Labelling of stem cells, assisted by these imaging techniques, has proven to be useful in establishing stem cell lineage for regenerative therapy of the oro-facial tissue complex. Novel labelling methods complementing imaging techniques have been pivotal in understanding craniofacial tissue development and regeneration. These stem cell tracking methods have the potential to facilitate the development of innovative cell-based therapies.

Successful Application of a Galanin-Coated Scaffold for Periodontal Regeneration

The nervous system exerts finely tuned control over all aspects of the life of an organism, including pain, sensation, growth, and development. Recent developments in tissue regeneration research have increasingly turned to small molecule peptides to tailor and augment the biological response following tissue loss or injury. In the present study, we have introduced the small molecule peptide galanin (GAL) as a novel scaffold-coating agent for the healing and regeneration of craniofacial tissues. Using immunohistochemistry, we detected GAL and GAL receptors in healthy periodontal tissues and in the proximity of blood vessels, while exposure to our periodontal disease regimen resulted in a downregulation of GAL. In a 3-dimensional bioreactor culture, GAL coating of collagen scaffolds promoted cell proliferation and matrix synthesis. Following subcutaneous implantation, GAL-coated scaffolds were associated with mineralized bone-like tissue deposits, which reacted positively for alizarin red and von Kossa, and demonstrated increased expression and protein levels of RUNX2, OCN, OSX, and iBSP. In contrast, the GAL receptor antagonist galantide blocked the effect of GAL on Runx2 expression and inhibited mineralization in our subcutaneous implantation model. Moreover, GAL coating promoted periodontal regeneration and a rescue of the periodontal defect generated in our periodontitis model mice. Together, these data demonstrate the efficacy of the neuropeptide GAL as a coating material for tissue regeneration. They are also suggestive of a novel role for neurogenic signaling pathways in craniofacial and periodontal regeneration.

Strategies of Prevascularization in Tissue Engineering and Regeneration of Craniofacial Tissues.

Craniofacial tissue defects caused by trauma, developmental malformation, or surgery are critical issues of high incidence, which are harmful to physical and psychological health. Transplantation of engineered tissues or biomaterials is a potential method to repair defects and regenerate the craniofacial tissues. Revascularization is essential to ensure the survival and regeneration of the grafts. Since microvessels play a critical role in blood circulation and substance exchange, the pre-establishment of the microvascular network in transplants provides a technical basis for the successful regeneration of the tissue defect. In this study, we reviewed the recent development of strategies and applications of prevascularization in tissue engineering and regeneration of craniofacial tissues. We focused on the cellular foundation of the in vitro prevascularized microvascular network, the cell source for prevascularization, and the strategies of prevascularization. Several key strategies, including coculture, microspheres, three-dimensional printing and microfluidics, and microscale technology, were summarized and the feasibility of these technologies in the clinical repair of craniofacial defects was discussed. Impact statement Prevascularization is an effective strategy to promote the survival and regeneration of the graft after transplantation. This review focuses on recent development in the technique of prevascularization and discusses the advantages and limits of current technologies. Furthermore, we summarized the progress in the repair of craniofacial tissue defect with prevascularization strategies. The development raised promising prospects of prevascularization strategies in the regeneration of cranial facial tissue defects in the future.

Translating Dental, Oral, and Craniofacial Regenerative Medicine Innovations to the Clinic through Interdisciplinary Commercial Translation Architecture

Few university-based regenerative medicine innovations in the dental, oral, and craniofacial (DOC) space have been commercialized and affected clinical practice in the United States. An analysis of the commercial translation literature and National Institute for Dental and Craniofacial Research’s (NIDCR’s) portfolio identified barriers to commercial translation of university-based DOC innovations. To overcome these barriers, the NIDCR established the Dental Oral Craniofacial Tissue Regeneration Consortium. We provide generalized strategies to inform readers how to bridge the “valley of death” and more effectively translate DOC technologies from the research laboratory or early stage company environment to clinical trials and bring needed innovations to the clinic. Three valleys of death are covered: 1) from basic science to translational development, 2) from translational technology validation to new company formation (or licensing to an existing company), and 3) from new company formation to scaling toward commercialization. An adapted phase-gate model is presented to inform DOC regenerative medicine teams how to involve regulatory, manufacturability, intellectual property, competitive assessments, business models, and commercially oriented funding mechanisms earlier in the translational development process. An Industrial Partners Program describes how to conduct market assessments, industry maps, business development processes, and industry relationship management methods to sustain commercial translation through the later-stage valley of death. Paramount to successfully implementing these methods is the coordination and collaboration of interdisciplinary teams around specific commercial translation goals and objectives. We also provide several case studies for translational projects with an emphasis on how they addressed DOC biomaterials for tissue regeneration within a rigorous commercial translation development environment. These generalized strategies and methods support innovations within a university-based and early stage company-based translational development process, traversing the many funding gaps in dental, oral, and craniofacial regenerative medicine innovations. Although the focus is on shepherding technologies through the US Food and Drug Administration, the approaches are applicable worldwide.

Exosomes derived from stem cells from apical papilla promote craniofacial soft tissue regeneration by enhancing Cdc42-mediated vascularization

BackgroundReconstruction of complex critical-size defects (CSD) in the craniofacial region is a major challenge, and soft tissue regeneration is crucial in determining the therapeutic outcomes of craniofacial CSD. Stem cells from apical papilla (SCAP) are neural crest-derived mesenchymal stem cells (MSCs) that are homologous to cells in craniofacial tissue and represent a promising source for craniofacial tissue regeneration. Exosomes, which contain compound bioactive compounds, are the key factors in stem cell paracrine action. However, the roles of exosomes derived from SCAP (SCAP-Exo) in tissue regeneration are not fully understood. Here, we explored the effects and underlying mechanisms of SCAP-Exo on CSD in maxillofacial soft tissue.MethodsSCAP-Exo were isolated and identified by transmission electron microscopy and nanoparticle tracking analysis. The effects of SCAP-Exo on wound healing and vascularization were detected by measuring the wound area and performing histological and immunofluorescence analysis on the palatal gingival CSD of mice. Real-time live-cell imaging and functional assays were used to assess the effects of SCAP-Exo on the biological functions of endothelial cells (ECs). Furthermore, the molecular mechanisms of SCAP-Exo-mediated EC angiogenesis in vitro were tested by immunofluorescence staining, Western blot, and pull-down assays. Finally, in vivo experiments were carried out to verify whether SCAP-Exo could affect vascularization and wound healing through cell division cycle 42 (Cdc42).ResultsWe found that SCAP-Exo promoted tissue regeneration of palatal gingival CSD by enhancing vascularization in the early phase in vivo and that SCAP-Exo improved the angiogenic capacity of ECs in vitro. Mechanistically, SCAP-Exo elevated cell migration by improving cytoskeletal reorganization of ECs via Cdc42 signalling. Furthermore, we revealed that SCAP-Exo transferred Cdc42 into the cytoplasm of ECs and that the Cdc42 protein could be reused directly by recipient ECs, which resulted in the activation of Cdc42-dependent filopodium formation and elevation in cell migration of ECs.ConclusionThis study demonstrated that SCAP-Exo had a superior effect on angiogenesis and effectively promoted craniofacial soft tissue regeneration. These data provide a new option for SCAP-Exo to be used in a cell-free approach to optimize tissue regeneration in the clinic.

Potential of Lyophilized Platelet Concentrates for Craniofacial Tissue Regenerative Therapies.

Objective: The use of platelet concentrates (PCs) in oral and maxillofacial surgery, periodontology, and craniofacial surgery has been reported. While PCs provide a rich reservoir of autologous bioactive growth factors for tissue regeneration, their drawbacks include lack of utility for long-term application, low elastic modulus and strength, and limited storage capability. These issues restrict their broader application. This review focuses on the lyophilization of PCs (LPCs) and how this processing approach affects their biological and mechanical properties for application as a bioactive scaffold for craniofacial tissue regeneration. Materials and Methods: A comprehensive search of five electronic databases, including Medline, PubMed, EMBASE, Web of Science, and Scopus, was conducted from 1946 until 2019 using a combination of search terms relating to this topic. Results: Ten manuscripts were identified as being relevant. The use of LPCs was mostly studied in in vitro and in vivo craniofacial bone regeneration models. Notably, one clinical study reported the utility of LPCs for guided bone regeneration prior to dental implant placement. Conclusions: Lyophilization can enhance the inherent characteristics of PCs and extends shelf-life, enable their use in emergency surgery, and improve storage and transportation capabilities. In light of this, further preclinical studies and clinical trials are required, as LPCs offer a potential approach for clinical application in craniofacial tissue regeneration.

Cranial Neural Crest Cells and Their Role in the Pathogenesis of Craniofacial Anomalies and Coronal Craniosynostosis.

Craniofacial anomalies are among the most common of birth defects. The pathogenesis of craniofacial anomalies frequently involves defects in the migration, proliferation, and fate of neural crest cells destined for the craniofacial skeleton. Genetic mutations causing deficient cranial neural crest migration and proliferation can result in Treacher Collins syndrome, Pierre Robin sequence, and cleft palate. Defects in post-migratory neural crest cells can result in pre- or post-ossification defects in the developing craniofacial skeleton and craniosynostosis (premature fusion of cranial bones/cranial sutures). The coronal suture is the most frequently fused suture in craniosynostosis syndromes. It exists as a biological boundary between the neural crest-derived frontal bone and paraxial mesoderm-derived parietal bone. The objective of this review is to frame our current understanding of neural crest cells in craniofacial development, craniofacial anomalies, and the pathogenesis of coronal craniosynostosis. We will also discuss novel approaches for advancing our knowledge and developing prevention and/or treatment strategies for craniofacial tissue regeneration and craniosynostosis.

Gene-Environment Interplay and MicroRNAs in Cleft Lip and Cleft Palate.

Cleft lip (CL) with/without cleft palate (CP) (hereafter CL/P) is the second most common congenital birth defect, affecting 7.94 to 9.92 children per 10,000 live births worldwide, followed by Down syndrome. An increasing number of genes have been identified as affecting susceptibility and/or as causative genes for CL/P in mouse genetic and chemically-induced CL and CP studies, as well as in human genome-wide association studies and linkage analysis. While marked progress has been made in the identification of genetic and environmental risk factors for CL/P, the interplays between these factors are not yet fully understood. This review aims to summarize our current knowledge of CL and CP from genetically engineered mouse models and environmental factors that have been studied in mice. Understanding the regulatory mechanism(s) of craniofacial development may not only advance our understanding of craniofacial developmental biology, but could also provide approaches for the prevention of birth defects and for tissue engineering in craniofacial tissue regeneration.

An engineered cell-laden adhesive hydrogel promotes craniofacial bone tissue regeneration in rats.

Cell-laden hydrogels are widely used in tissue engineering and regenerative medicine. However, many of these hydrogels are not optimized for use in the oral environment, where they are exposed to blood and saliva. To address these challenges, we engineered an alginate-based adhesive, photocrosslinkable, and osteoconductive hydrogel biomaterial (AdhHG) with tunable mechanical properties. The engineered hydrogel was used as an injectable mesenchymal stem cell (MSC) delivery vehicle for craniofacial bone tissue engineering applications. Subcutaneous implantation in mice confirmed the biodegradability, biocompatibility, and osteoconductivity of the hydrogel. In a well-established rat peri-implantitis model, application of the adhesive hydrogel encapsulating gingival mesenchymal stem cells (GMSCs) resulted in complete bone regeneration around ailing dental implants with peri-implant bone loss. Together, we have developed a distinct bioinspired adhesive hydrogel with tunable mechanical properties and biodegradability that effectively delivers patient-derived dental-derived MSCs. The hydrogel is photocrosslinkable and, due to the presence of MSC aggregates and hydroxyapatite microparticles, promotes bone regeneration for craniofacial tissue engineering applications.