Field Of Neural Tissue Engineering Research Articles

A 3D printable gelatin methacryloyl/chitosan hydrogel assembled with conductive PEDOT for neural tissue engineering

In neural tissue engineering, biomaterial scaffolds that have high conductivity and customized structures are crucial in promoting nerve regeneration. Poly(3,4-ethylenedioxythiophene) (PEDOT) has emerged as a promising conductive polymer with excellent chemical stability and biocompatibility. However, traditional three-dimensional (3D) printing of PEDOT-based conductive scaffolds faces challenges in limited printing resolution, poor solubility, and brittleness of conductive materials. Herein, digital light processing (DLP) printing was used to fabricate complex hydrogel structures using gelatin methacryloyl (GelMA) and chitosan (CS) while incorporating PEDOT nanoparticles through interfacial polymerization to create conducting pathways within a hydrogel structure. The integration of PEDOT significantly enhanced the electrical conductivity and mechanical properties of the GelMA/CS hydrogel while preserving printed details. The GelMA/CS-PEDOT hydrogel promoted cell proliferation and facilitated axon outgrowth of PC12 cells and Schwann cells during in vitro culture. Moreover, in vitro direct current electrical stimulation promoted axon elongation of PC12 cells cultured on a conductive substrate. In vivo studies used a conductive nerve conduit to repair a 10-mm rat sciatic nerve defect, validating the efficacy of GelMA/CS-PEDOT scaffold in peripheral nerve injury repair. These findings highlight the significant potential of conductive GelMA/CS-PEDOT hydrogel in the field of neural tissue engineering.

Potential of dental pulp stem cells and their products in promoting peripheral nerve regeneration and their future applications

Peripheral nerve injury (PNI) seriously affects people’s quality of life. Stem cell therapy is considered a promising new option for the clinical treatment of PNI. Dental stem cells, particularly dental pulp stem cells (DPSCs), are adult pluripotent stem cells derived from the neuroectoderm. DPSCs have significant potential in the field of neural tissue engineering due to their numerous advantages, such as easy isolation, multidifferentiation potential, low immunogenicity, and low transplant rejection rate. DPSCs are extensively used in tissue engineering and regenerative medicine, including for the treatment of sciatic nerve injury, facial nerve injury, spinal cord injury, and other neurodegenerative diseases. This article reviews research related to DPSCs and their advantages in treating PNI, aiming to summarize the therapeutic potential of DPSCs for PNI and the underlying mechanisms and providing valuable guidance and a foundation for future research.

MicroRNA-584-5p/RUNX family transcription factor 2 axis mediates hypoxia-induced osteogenic differentiation of periosteal stem cells.

The hypoxic environment during bone healing is important in regulating the differentiation of periosteal stem cells (PSCs) into osteoblasts or chondrocytes; however, the underlying mechanisms remain unclear. To determine the effect of hypoxia on PSCs, and the expression of microRNA-584-5p (miR-584-5p) and RUNX family transcription factor 2 (RUNX2) in PSCs was modulated to explore the impact of the miR-584-5p/RUNX2 axis on hypoxia-induced osteogenic differentiation of PSCs. In this study, we isolated primary mouse PSCs and stimulated them with hypoxia, and the characteristics and functional genes related to PSC osteogenic differentiation were assessed. Constructs expressing miR-584-5p and RUNX2 were established to determine PSC osteogenic differentiation. Hypoxic stimulation induced PSC osteogenic differentiation and significantly increased calcified nodules, intracellular calcium ion levels, and alkaline phosphatase (ALP) activity in PSCs. Osteogenic differentiation-related factors such as RUNX2, bone morphogenetic protein 2, hypoxia-inducible factor 1-alpha, and ALP were upregulated; in contrast, miR-584-5p was downregulated in these cells. Furthermore, upregulation of miR-584-5p significantly inhibited RUNX2 expression and hypoxia-induced PSC osteogenic differentiation. RUNX2 was the target gene of miR-584-5p, antagonizing miR-584-5p inhibition in hypoxia-induced PSC osteogenic differentiation. Our study showed that the interaction of miR-584-5p and RUNX2 could mediate PSC osteogenic differentiation induced by hypoxia.

Design of IKVAV peptide/gold nanoparticle decorated, micro/nano-channeled PCL/PLGA film scaffolds for neuronal differentiation and neurite outgrowth

In the field of neural tissue engineering, intensive efforts are being made to develop tissue scaffolds that can support an effective functional recovery and neural development by guiding damaged axons and neurites. Micro/nano-channeled conductive biomaterials are considered a promising approach for repairing the injured neural tissues. Many studies have demonstrated that the micro/nano-channels and aligned nanofibers could guide the neurites to extend along the direction of alignment. However, an ideal biocompatible scaffold containing conductive arrays that could promote effective neural stem cell differentiation and development, and also stimulate high neurite guidance has not been fully developed. In the current study, we aimed to fabricate micro/nano-channeled polycaprolactone (PCL)/Poly-d,l-lactic-co-glycolic acid (PLGA) hybrid film scaffolds, decorate their surfaces with IKVAV pentapeptide/gold nanoparticles (AuNPs), and investigate the behavior of PC12 cells and neural stem cells (NSCs) on the developed biomaterial under static/bioreactor conditions. Here we show that channeled groups decorated with AuNPs highly promote neurite outgrowth and neuronal differentiation along linear lines in the presence of electrical stimulation, compared with the polypyrrole (PPy) coating, which has been used traditionally for many years. Hopefully, this newly developed channeled scaffold structure (PCL/PLGA-AuNPs-IKVAV) could help to support long-distance axonal regeneration and neuronal development after different neural damages.

Accelerating Neurite Growth and Directing Neuronal Connections Constrained by 3D Porous Microtubes.

Investigation of neural growth and connection is crucial in the field of neural tissue engineering. Here, using a femtosecond laser direct writing (fs-DLW) technique, we propose a directionally aligned porous microtube array as a culture system for accelerating the growth of neurons and directing the connection of neurites. These microtubes exhibited an unprecedented guidance effect toward the outgrowth of primary embryonic rat hippocampal neurons, with a wrap resembling the myelin sheaths of neurons. The speed of neurite growth inside these microtubes was significantly faster than that outside these microtubes. We also achieved selective/directing connection of neural networks inside the magnetic microtubes via precise microtube delivery to a gap between two neural clusters. This work not only proposes a powerful microtube platform for accelerated growth of neurons but also offers a new idea for constructing biological neural circuits by arranging the size, location, and pattern of microtubes.

The optimal electrical stimulation for neural differentiation of conjunctiva mesenchymal stem cells.

The combination of biomaterial conductive scaffolds and electrical stimulation (ES) dramatically promotes stem cell differentiation into electro-responsive cells like neural cells. In this study, we aimed to fabricate PCL/PPY nanofiber scaffolds through the electrospinning method and investigate the effect of ES duration on neural differentiation of Conjunctiva Mesenchymal Stem Cells (CJMSCs). The topography of the fabricated scaffold was characterized using SEM and TEM microscopy, and its mechanical and other properties were determined by tensile, TGA, FTIR, and Contact angle tests. CJMSCs were seeded on the scaffolds and then subjected to electrical current (115 V m-1 at 100 Hz) with durations of 1, 3, and 7 min for 3 days. Then the effect of nanofiber scaffold and electrical currents on cell viability and expression of neural marker genes (Nestin, β-tubulin, MAP-2) was investigated by MTT assay and qPCR analysis. Our results revealed the good biocompatibility of the PCL-PPy nanofiber scaffold, and according to q-PCR results, the electrical stimulation of 1 min day-1 for 3 days can induce neural differentiation of CJMSCs as indicated by the fold change of gene expression of Nestin (~127), B-tubulin (~30), and MAP-2 (~52). This study emphasizes that the utilization of an electrically conductive nanofibrous scaffold in conjunction with electrical current has potential applications in the field of neural tissue engineering.

Scaffolds with anisotropic structure for neural tissue engineering

Nervous system injuries remain a great challenge due to limited natural tissue regeneration capabilities. Neural tissue engineering has been regarded as a promising approach for repairing nerve defects, which utilizes external biomaterial scaffolds to allow cells to migrate to the injury site and repair the tissue. Particularly, scaffolds with anisotropic structures biomimicking the native extracellular matrix (ECM) can effectively guide neural orientation and reconnection. Here, the advancements of scaffolds with anisotropic structures in the field of neural tissue engineering are presented. The fabrication strategies of scaffolds with anisotropic structures and their effects in vitro and in vivo are highlighted. We also discuss the challenges and provide a perspective of this field.

Sustainable release of nerve growth factor for peripheral nerve regeneration using nerve conduits laden with Bioconjugated hyaluronic acid-chitosan hydrogel

Peripheral nerve regeneration following injury is often incomplete, resulting in significant personal and socioeconomic costs. Nerve conduits have been developed to complement or replace autologous nerve grafts. In this study, repairing injured peripheral nerves in rats is investigated by fabricating poly(D, l-lactic acid) (PDLLA)/β-tricalcium phosphate (β-TCP) nerve conduits filled by injectable chitosan (CS)-hyaluronic acid (HA) hydrogels featuring sustained release of nerve growth factor (NGF). All CS-HA hydrogels have porosities higher than 80%. The mass loss of the CS-HA hydrogels reaches 70% in 8 weeks. In vitro experiments suggest that the CS-HA/NGF hydrogels are suitable for the adhesion, spreading, and differentiation of neuronal cells, which also allow for sustained release of NGF. In vivo studies on the repair of 10-mm sciatic nerve defects in rats are used to assess nerve repair rate through walking track analysis, gastrocnemius weight analysis, and histological analysis. It is shown that the PDLLA/β-TCP nerve conduits laden with CS-HA/NGF hydrogels have a significant positive effect on the axon regeneration and myelination, compared to the PDLLA/β-TCP hollow nerve conduits and the autograft group. These results show that the CS-HA/NGF injectable hydrogel can effectively promote nerve regeneration making it a good candidate in the field of neural tissue engineering.

Bifunctional conducting polymer matrices with antibacterial and neuroprotective effects

Current trends in the field of neural tissue engineering include the design of advanced biomaterials combining excellent electrochemical performance with versatile biological characteristics. The purpose of this work was to develop an antibacterial and neuroprotective coating based on a conducting polymer – poly(3,4-ethylenedioxypyrrole) (PEDOP), loaded with an antibiotic agent – tetracycline (Tc). Employing an electrochemical technique to immobilize Tc within a growing polymer matrix allowed to fabricate robust PEDOP/Tc coatings with a high charge storage capacity (63.65 ± 6.05 mC/cm2), drug release efficiency (629.4 µg/cm2 ± 62.7 µg/cm2), and low charge transfer resistance (2.4 ± 0.1 kΩ), able to deliver a stable electrical signal. PEDOP/Tc were found to exhibit strong antimicrobial effects against Gram-negative bacteria Escherichia coli, expressed through negligible adhesion, reduction in viability, and a characteristic elongation of bacterial cells. Cytocompatibility and neuroprotective effects were evaluated using a rat neuroblastoma B35 cell line, and were analyzed using MTT, cell cycle, and Annexin-V apoptosis assays. The presence of Tc was found to enhance neural cell viability and neurite outgrowth. The results confirmed that PEDOP/Tc can serve as an efficient neural electrode coating able to enhance charge transfer, as well as to exhibit bifunctional biological characteristics, different for eukaryotic and prokaryotic cells.

Biocompatibility evaluation of a 3D-bioprinted alginate-GelMA-bacteria nanocellulose (BNC) scaffold laden with oriented-growth RSC96 cells

Peripheral nerve injury can cause various degrees of damage to the morphological structure and physiological function of the peripheral nerve. At present, compared with “gold standard” autologous nerve transplantation, tissue engineering has certain potential for regeneration and growth; however, achieving oriented guidance is still a challenge. In this study, we used 3D bioprinting to construct a nerve scaffold of RSC96 cells wrapped in sodium alginate/gelatin methacrylate (GelMA)/bacterial nanocellulose (BNC) hydrogel. The 5% sodium alginate+5% GelMA+0.3% BNC group had the thinnest lines among all groups after printing, indicating that the inherent shape of the scaffold could be maintained after adding BNC. Physical and chemical property testing (Fourier transform infrared, rheometer, conductivity, and compression modulus) showed that the 5% alginate+5% GelMA+0.3% BNC group had better mechanical and rheological properties. Live/dead cell staining showed that no mass cell death was observed on days 1, 3, 5, and 7 after printing. In the 5% alginate+5% GelMA group, the cells grew and formed linear connections in the scaffold. This phenomenon was more obvious in the 5% alginate+5% GelMA+0.3% BNC group. In the 5% alginate+5% GelMA+0.3% BNC group, S-100β immunofluorescence staining and cytoskeleton staining showed oriented growth. Polymerase chain reaction (PCR) array results showed that mRNA levels of related neurofactors ASCL1, POU3F3, NEUROG1, DLL1, NOTCH1 and ERBB2 in the 5%GelMA+0.3%BNC group were higher than those of other groups. Four weeks after implantation in nude mice, RSC96 cells grew and proliferated well, blood vessels grew, and S-100β immunofluorescence was positive. These results indicate that a 3D-bioprinted sodium alginate/GelMA/BNC composite scaffold can improve cell-oriented growth, adhesion and the expression of related factors. This 3D-bioprinted composite scaffold has good biocompatibility and is expected to become a new type of scaffold material in the field of neural tissue engineering.

Properties Regulation and Biological Applications of Decellularized Peripheral Nerve Matrix Hydrogel.

Decellularized peripheral nerve matrix hydrogel (DNM-G) has drawn increasing attention in the field of neural tissue engineering, owing to its high tissue-specific bioactivity, drug/cell delivery capability, and multifunctional processability. However, the mechanisms and influencing factors of DNM-G formation have been rarely reported. To enable potential biological applications, the relationship between gelation conditions (including digestion time and gel concentration) and mechanical properties/stability (sol-gel transition temperature, gelation time, nanotopology, and storage modulus) of the DNM-G were systematically investigated in this study. The adequate-digested decellularized nerve matrix solution exhibited higher mechanical property, shorter gelation time, and a lower gelation temperature. A noteworthy increase of β-sheet proportion was identified through Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD) characterizations, which suggested the possible major secondary structure formation during the phase transition. Besides, the DNM-G degraded fast that over 70% mass loss was noted after 4 weeks when immersing in PBS. A natural cross-linking agent, genipin, was gently introduced into DNM-G to enhance its mechanical properties and stability without changing its microstructure and biological performance. As a prefabricated scaffold, DNM-G remarkably increased the length and penetration depth of dorsal root ganglion (DRG) neurites compared to collagen gel. Furthermore, the DNM-G promoted the myelination and facilitated the formation of the morphological neural network. Finally, we demonstrated the feasibility of applying DNM-G in support-free extrusion-based 3D printing. Overall, the mechanical and biological performance of DNM-G can be manipulated by tuning the processing parameters, which is key to the versatile applications of DNM-G in regenerative medicine.

3D Bioprinting of Neural Tissues.

The human nervous system is a remarkably complex physiological network that is inherently challenging to study because of obstacles to acquiring primary samples. Animal models offer powerful alternatives to study nervous system development, diseases, and regenerative processes, however, they are unable to address some species-specific features of the human nervous system. In vitro models of the human nervous system have expanded in prevalence and sophistication, but still require further advances to better recapitulate microenvironmental and cellular features. The field of neural tissue engineering (TE) is rapidly adopting new technologies that enable scientists to precisely control in vitro culture conditions and to better model nervous system formation, function, and repair. 3D bioprinting is one of the major TE technologies that utilizes biocompatible hydrogels to create precisely patterned scaffolds, designed to enhance cellular responses. This review focuses on the applications of 3D bioprinting in the field of neural TE. Important design parameters are considered when bioprinting neural stem cells are discussed. The emergence of various bioprinted in vitro platforms are also reviewed for developmental and disease modeling and drug screening applications within the central and peripheral nervous systems, as well as their use as implants for in vivo regenerative therapies.

Nanostructured scaffolds based on bioresorbable polymers and graphene oxide induce the aligned migration and accelerate the neuronal differentiation of neural stem cells

Within the field of neural tissue engineering, there is a huge need for the development of materials that promote the adhesion, aligned migration and differentiation of stem cells into neuronal and supportive glial cells. In this study, we have fabricated bioresorbable elastomeric scaffolds combining an ordered nanopatterned topography together with a surface functionalization with graphene oxide (GO) in mild conditions. These scaffolds allowed the attachment of murine neural stem cells (NSCs) without the need of any further coating of its surface with extracellular matrix adhesion proteins. The NSCs were able to give rise to both immature neurons and supporting glial cells over the nanostructured scaffolds in vitro, promoting their aligned migration in cell clusters following the nanostructured grooves. This system has the potential to reestablish spatially oriented neural precursor cell connectivity, constituting a promising tool for future cellular therapy including nerve tissue regeneration.

Nebulized jet-based printing of bio-electrical scaffolds for neural tissue engineering: a feasibility study

In this paper we investigate the application of a direct writing technique for printing conductive patterns onto a biocompatible electrospun-pyrolysed carbon-fibre-based substrate. The result is a first study towards the production of bio-electrical scaffolds that could be used to enhance the promotion of efficient connections among neurons for in vitro studies in the field of neural tissue engineering. An electrospinning process is employed for production of the materials derived from the precursor polyacrylonitrile, in which the embedding of carbon nanotubes (CNTs) is also investigated. Subsequently, the methodology of research into suitable parameters for the printed electronics, using a commercial silver nanoparticle (Øavg,particle size ∼ 100 nm) ink, is described. The results show values of 2 Ω cm for the resistivity of the carbon-fibre materials and conductive printed lines of resistance ∼50 Ω on glass and less than ∼140 Ω on carbon-fibre samples. Biocompatibility results demonstrate the possibility of using electrospun-pyrolysed mats, also with embedded CNTs, as potential neural substrates for spatially localized electrical stimulation across a tissue. In addition, the data concerning the potential toxicity of silver suspensions are in accordance with the literature, showing a dose-dependent behaviour. This work is a pioneering feasibility study of the use of the flexible and versatile printed electronic approach, combined with engineered biocompatible substrates, to realize integrated bio-electrical scaffolds for in vitro neural tissue engineering applications.

Tissue Response to Neural Implants: The Use of Model Systems Toward New Design Solutions of Implantable Microelectrodes.

The development of implantable neuroelectrodes is advancing rapidly as these tools are becoming increasingly ubiquitous in clinical practice, especially for the treatment of traumatic and neurodegenerative disorders. Electrodes have been exploited in a wide number of neural interface devices, such as deep brain stimulation, which is one of the most successful therapies with proven efficacy in the treatment of diseases like Parkinson or epilepsy. However, one of the main caveats related to the clinical application of electrodes is the nervous tissue response at the injury site, characterized by a cascade of inflammatory events, which culminate in chronic inflammation, and, in turn, result in the failure of the implant over extended periods of time. To overcome current limitations of the most widespread macroelectrode based systems, new design strategies and the development of innovative materials with superior biocompatibility characteristics are currently being investigated. This review describes the current state of the art of in vitro, ex vivo, and in vivo models available for the study of neural tissue response to implantable microelectrodes. We particularly highlight new models with increased complexity that closely mimic in vivo scenarios and that can serve as promising alternatives to animal studies for investigation of microelectrodes in neural tissues. Additionally, we also express our view on the impact of the progress in the field of neural tissue engineering on neural implant research.

Scaffold-mediated sequential drug/gene delivery to promote nerve regeneration and remyelination following traumatic nerve injuries.

Neural tissue regeneration following traumatic injuries is often subpar. As a result, the field of neural tissue engineering has evolved to find therapeutic interventions and has seen promising outcomes. However, robust nerve and myelin regeneration remain elusive. One possible reason may be the fact that tissue regeneration often follows a complex sequence of events in a temporally-controlled manner. Although several other fields of tissue engineering have begun to recognise the importance of delivering two or more biomolecules sequentially for more complete tissue regeneration, such serial delivery of biomolecules in neural tissue engineering remains limited. This review aims to highlight the need for sequential delivery to enhance nerve regeneration and remyelination after traumatic injuries in the central nervous system, using spinal cord injuries as an example. In addition, possible methods to attain temporally-controlled drug/gene delivery are also discussed for effective neural tissue regeneration.

Silicon nanowires enhanced proliferation and neuronal differentiation of neural stem cell with vertically surface microenvironment

Owing to its biocompatibility, noncytotoxicity, biodegradability and three-dimensional structure, vertically silicon nanowires (SiNWs) arrays are a promising scaffold material for tissue engineering, regenerative medicine and relevant medical applications. Recently, its osteogenic differentiation effects, reorganization of cytoskeleton and regulation of the fate on stem cells have been demonstrated. However, it still remains unknown whether SiNWs arrays could affect the proliferation and neuronal differentiation of neural stem cells (NSCs) or not. In the present study, we have employed vertically aligned SiNWs arrays as culture systems for NSCs and proved that the scaffold material could promote the proliferation and neuronal differentiation of NSCs while maintaining excellent cell viability and stemness. Immunofluorescence imaging analysis, Western blot and RT-PCR results reveal that NSCs proliferation and neuronal differentiation efficiency on SiNWs arrays are significant greater than that on silicon wafers. These results implicate SiNWs arrays could offer a powerful platform for NSCs research and NSCs-based therapy in the field of neural tissue engineering.

Progress and perspectives of neural tissue engineering.

Traumatic injuries to the nervous system lead to a common clinical problem with a quite high incidence and affect the patient’s quality of life. Based on a major challenge not yet addressed by current therapeutic interventions for these diseases, a novel promising field of neural tissue engineering has emerged, grown, and attracted increasing interest. This review provides a brief summary of the recent progress in the field, especially in combination with the research experience of the author’s group. Several important aspects related to tissue engineered nerves, including the theory on their construction, translation into the clinic, improvements in fabrication technologies, and the formation of a regenerative environment, are delineated and discussed.Furthermore, potential research directions for the future development of neural tissue engineering are suggested.

Neural tissue engineering options for peripheral nerve regeneration

Tissue engineered nerve grafts (TENGs) have emerged as a potential alternative to autologous nerve grafts, the gold standard for peripheral nerve repair. Typically, TENGs are composed of a biomaterial-based template that incorporates biochemical cues. A number of TENGs have been used experimentally to bridge long peripheral nerve gaps in various animal models, where the desired outcome is nerve tissue regeneration and functional recovery. So far, the translation of TENGs to the clinic for use in humans has met with a certain degree of success. In order to optimize the TENG design and further approach the matching of TENGs with autologous nerve grafts, many new cues, beyond the traditional ones, will have to be integrated into TENGs. Furthermore, there is a strong requirement for monitoring the real-time dynamic information related to the construction of TENGs. The aim of this opinion paper is to specifically and critically describe the latest advances in the field of neural tissue engineering for peripheral nerve regeneration. Here we delineate new attempts in the design of template (or scaffold) materials, especially in the context of biocompatibility, the choice and handling of support cells, and growth factor release systems. We further discuss the significance of RNAi for peripheral nerve regeneration, anticipate the potential application of RNAi reagents for TENGs, and speculate on the possible contributions of additional elements, including angiogenesis, electrical stimulation, molecular inflammatory mediators, bioactive peptides, antioxidant reagents, and cultured biological constructs, to TENGs. Finally, we consider that a diverse array of physicochemical and biological cues must be orchestrated within a TENG to create a self-consistent coordinated system with a close proximity to the regenerative microenvironment of the peripheral nervous system.

Nanomaterials design and tests for neural tissue engineering

Nanostructured scaffolds recently showed great promise in tissue engineering: nanomaterials can be tailored at the molecular level and scaffold morphology may more closely resemble features of extracellular matrix components in terms of porosity, framing and biofunctionalities. As a consequence, both biomechanical properties of scaffold microenvironments and biomaterial-protein interactions can be tuned, allowing for improved transplanted cell engraftment and better controlled diffusion of drugs. Easier said than done, a nanotech-based regenerative approach encompasses different fields of know-how, ranging from in silico simulations, nanomaterial synthesis and characterization at the nano-, micro- and mesoscales to random library screening methods (e.g. phage display), in vitro cellular-based experiments and validation in animal models of the target injury. All of these steps of the "assembly line" of nanostructured scaffolds are tightly interconnected both in their standard analysis techniques and in their most recent breakthroughs: indeed their efforts have to jointly provide the deepest possible analyses of the diverse facets of the challenging field of neural tissue engineering. The purpose of this review is therefore to provide a critical overview of the recent advances in and drawbacks and potential of each mentioned field, contributing to the realization of effective nanotech-based therapies for the regeneration of peripheral nerve transections, spinal cord injuries and brain traumatic injuries. Far from being the ultimate overview of such a number of topics, the reader will acknowledge the intrinsic complexity of the goal of nanotech tissue engineering for a conscious approach to the development of a regenerative therapy and, by deciphering the thread connecting all steps of the research, will gain the necessary view of its tremendous potential if each piece of stone is correctly placed to work synergically in this impressive mosaic.