A comprehensive conceptual and computational dynamics framework for autonomous regeneration systems

This paper presents a new conceptual and computational dynamics framework for damage detection and regeneration in multicellular structures similar to living animals. The model uniquely achieves complete and accurate regeneration from any damage anywhere in the system. We demonstrated the efficacy of the proposed framework on an artificial organism consisting of three tissue structures corresponding to the head, body and tail of a worm. Each structure consists of a stem cell surrounded by a tissue of differentiated cells. We represent a tissue as an Auto-Associative Neural Network (AANN) with local interactions and stem cells as a self-repair network with long-range interactions. We also propose another new concept, Information Field which is a mathematical abstraction over traditional components of tissues, to keep minimum pattern information of the tissue structures to be accessed by stem cells in extreme cases of damage. Through entropy, a measure of communication between a stem cell and differentiated cells, stem cells monitor the tissue pattern integrity, violation of which triggers damage detection and tissue repair. Stem cell network monitors its state and invokes stem cell repair in the case of stem cell damage. The model accomplishes regeneration at two levels: In the first level, damaged tissues with intact stem cells regenerate themselves. Here, stem cell identifies entropy change and finds the damage and regenerates the tissue in collaboration with the AANN. In the second level, involving missing whole tissues and stem cells, the remaining stem cell(s) access the information field to restore the stem cell network and regenerate missing tissues. In the case of partial tissue damage with missing stem cells, the two levels collaborate to accurately restore the stem cell network and tissues. This comprehensive hypothetical framework offers a new way to conceptualise regeneration for better understanding the regeneration processes in living systems. It could also be useful in biology for regenerative medicine and in engineering for building self-repairing biobots

A comprehensive conceptual and computational dynamics framework for autonomous regeneration of form and function in biological organisms

In biology, regeneration is a mysterious phenomenon that has inspired self-repairing systems, robots, and biobots. It is a collective computational process whereby cells communicate to achieve an anatomical set point and restore original function in regenerated tissue or the whole organism. Despite decades of research, the mechanisms involved in this process are still poorly understood. Likewise, the current algorithms are insufficient to overcome this knowledge barrier and enable advances in regenerative medicine, synthetic biology, and living machines/biobots. We propose a comprehensive conceptual framework for the engine of regeneration with hypotheses for the mechanisms and algorithms of stem cell-mediated regeneration that enables a system like the planarian flatworm to fully restore anatomical (form) and bioelectric (function) homeostasis from any small- or large-scale damage. The framework extends the available regeneration knowledge with novel hypotheses to propose collective intelligent self-repair machines, with multi-level feedback neural control systems, driven by somatic and stem cells. We computationally implemented the framework to demonstrate the robust recovery of both anatomical and bioelectric homeostasis in an worm that, in a simple way, resembles the planarian. In the absence of complete regeneration knowledge, the framework contributes to understanding and generating hypotheses for stem cell mediated form and function regeneration which may help advance regenerative medicine and synthetic biology. Further, as our framework is a bio-inspired and bio-computing self-repair machine, it may be useful for building self-repair robots/biobots and artificial self-repair systems

Autonomous self-repair systems : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Regeneration is an important and wonderful phenomenon in nature and plays a key role in living organisms that are capable of recovery from trivial to serious injury to reclaim a fully functional state and pattern/anatomical homeostasis (equilibrium). Studying regeneration can help develop hypotheses for understanding regenerative mechanisms along with advancing synthetic biology for regenerative medicine and development of cancer and anti-ageing drugs. Further, it can contribute to nature-inspired computing for self-repair in other fields. However, despite decades of study, what possible mechanisms and algorithms are used in the regeneration process remain an open question. Therefore, the main goal of this thesis is to propose a comprehensive hypothetical conceptual framework with possible mechanisms and algorithms of biological regeneration that mimics the observed features of regeneration in living organisms and achieves body-wide immortality, similar to the planarian flatworm, about 20mm long and 3mm wide, living in both saltwater and freshwater. This is a problem of collective decision making by the cells in an organism to achieve the high-level goal of returning to normality of both anatomical and functional homeostasis. To fulfil this goal, the proposed framework contains three sub-frameworks corresponding to three main objectives of the thesis: self-regeneration or self-repair (anatomical homeostasis) of a simple in silico tissue and a whole organism consisting of these tissues based on simplified formats of cellular communication, and an extension to more realistic bioelectric communication for restoring both anatomical and bioelectric homeostasis. The first objective is to develop a simple tissue model that regenerates autonomously after damage. Accordingly, we present a computational framework for an autonomous self-repair system that allows for sensing, detecting and regenerating an artificial (in silico) circular tissue containing thousands of cells. This system consists of two sub-models: Global Sensing and Local Sensing that collaborate to sense and repair diverse damages. It is largely a neural system with a perceptron (binary) network performing tissue computations. The results showed that the system is robust and efficient in damage detection and accurate regeneration. The second objective is to extend the simple circular tissue model to other geometric shapes and assemble them into a small virtual organism that regenerates similar to the body-wide immortality of the planarian flatworm. Accordingly, we proposed a computational framework extending the tissue repair framework developed in Objective 1 to model whole organism regeneration that implemented algorithms and mechanisms to achieve accurate and complete regeneration in an (in silico) worm-like organism. The system consists of two levels: tissue and organism levels that integrate to recognise and recover from any damage, even extreme damage cases. The tissue level consists of three tissue repair models for head, body and tail. The organism level connects the tissues together to form the worm. The two levels form an integrated neural feedback control system with perceptron (binary) for tissue computing and linear neural networks for organism-level computing. Our simulation results showed that the framework is very robust in returning the system to the normal state after any small or large scale damage. The last objective is to extend the whole organism regeneration framework developed in Objective 2 by incorporating bioelectricity as the format of communication between cells to make the model better resemble living organisms and to restore not only anatomy but also basic functionality such as restoring body-wide bioelectric pattern needed for physiological functioning in living systems. We greatly extended the second framework by conceptualising and modelling mechanisms and algorithms that mimicked both the pattern and function restoration observed in living organisms and implemented it on the same artificial (in silico) organism developed in Objective 2 but with greater realism of the anatomical structure. This proposed framework consists of three levels that collaborate to fully regenerate the anatomical pattern and maintain bioelectric homeostasis in the in silico worm-like organism. These three levels represent tissue and organism models for regeneration and body-wide bioelectric model for restoring bioelectric homeostasis, respectively. They extend the previous neural feedback control system to integrate another (3rd) level, bioelectric homeostasis. Our simulations showed that the system maintains and restores bioelectric homeostasis accurately under random perturbations of bioelectric status under no damage conditions. It is also very robust and plastic in restoring the system to the normal anatomical pattern and bioelectric homeostasis after any type of damage. Our framework robustly achieves some observations of extreme regeneration of planaria like body-wide immortality. It could also be helpful in engineering for building self-repair robots, biobots and artificial self-repair systems

Recent Progress and Potential Biomedical Applications of Electrospun Nanofibers in Regeneration of Tissues and Organs

Electrospun techniques are promising and flexible technologies to fabricate ultrafine fiber/nanofiber materials from diverse materials with unique characteristics under optimum conditions. These fabricated fibers/nanofibers via electrospinning can be easily assembled into several shapes of three-dimensional (3D) structures and can be combined with other nanomaterials. Therefore, electrospun nanofibers, with their structural and functional advantages, have gained considerable attention from scientific communities as suitable candidates in biomedical fields, such as the regeneration of tissues and organs, where they can mimic the network structure of collagen fiber in its natural extracellular matrix(es). Due to these special features, electrospinning has been revolutionized as a successful technique to fabricate such nanomaterials from polymer media. Therefore, this review reports on recent progress in electrospun nanofibers and their applications in various biomedical fields, such as bone cell proliferation, nerve regeneration, and vascular tissue, and skin tissue, engineering. The functionalization of the fabricated electrospun nanofibers with different materials furnishes them with promising properties to enhance their employment in various fields of biomedical applications. Finally, we highlight the challenges and outlooks to improve and enhance the application of electrospun nanofibers in these applications

Microring stereo sensor model using Kerr–Vernier effect for bio-cell sensor and communication

In this paper, a micro-stereo sensor is proposed using two-identical Panda-ring resonators, which are coupled by jointed drop ports. When light from the identical coherent sources is fed into the system via the input ports, the coupling outputs are obtained at the drop port at the resonant condition. These are mixed signals in the form of stereo signals. By using different input power between the right and left systems, the phase difference generated by the Kerr-Effect in the non-linear medium leads to the shift in the coupling outputs. The shift in the center wavelength is the primary measurement of interest along with coupling crosstalk signals that are also visible at the output. The measurement self-calibration of the two channels is confirmed by the mixed channel signals. In the manipulation, the crosstalk signals can be used to interpret the cross-communication of bio-cells. The crosstalk results have shown the optical crosstalks of ∼2.0 and ∼2.5 dB are calculated and obtained, respectively. The stereo sensor sensitivity of ∼5.70 nmW−1is noted

Dynamic nanostructured scaffolds as advanced biomaterials

Growing replacement tissues and organs in the laboratory will revolutionise healthcare; however, the maturation of cells into functional tissue constructs requires the controlled presentation of biochemical factors within a mechanically suitable scaffold. In nature, the presentation of such signals is provided through factors and structures existent within the nanoarchitecture of the extracellular matrix (ECM); therefore, in tissue engineering there is significant need to develop dynamic advanced artificial tissue constructs capable of mimicking the complexities of the native ECM. The requirement for bioactive, innervated constructs that contain biologically relevant signals delivered through tuneable mechanisms has yet to be achieved. One approach to address this key-challenge is offered through bioprinting, which allows for the controlled spatial distribution of bioinks containing cells, structures and signals within a single printed construct. However, currently bioprinting applications are severely limited by bioink function - with the majority of bioinks either lacking sufficient mechanical properties or biochemical signalling. Therefore, there is a key need to develop bioinks which adequately mimic the native ECM on a nanostructured, chemical level - particularly in establishing effective control over cell fate and tissue innervation. Tissue composition and extracellular signalling varies substantially between tissue-types, and therefore, advanced approaches that allow for ease of mechanical and biological tuneability through modular mechanisms would provide a practical avenue for bioink development. Self-assembling peptides (SAPs) are a unique class of biomaterials capable of spontaneously forming simple biomimetic structures which entangle to form highly hydrated, bioactive networks with favourable conditions for cell maturation. These biomaterials are easily tuned through modification of amino acid sequence, enabling tailored control over biochemical signalling between cells and scaffold. This provides the ability to artificially replicate natural signalling in a controlled manner - bringing about desired cell behaviour. Using these peptides, a variety of synergistic ECM-protein analogues have been developed, including Fmoc-FRGDF containing fibronectin's attachment motif RGD, and Fmoc-DIKAV, containing laminin's attachment motif IKVAV. Fmoc-SAPs possess the ability to be further functionalised through macromolecule addition, allowing for the presentation of charged, developmentally or structurally-important macromolecules on the surface of peptide fibrils. These macromolecules can integrate with the peptide networks, facilitating additional signalling and allowing for mechanical tunability. Here, we take advantage of these properties to develop an advanced and dynamic bioink for bioprinting applications. Initially, material enhancement is investigated through development of multi-sequence scaffolds. Specifically, Fmoc-FRGDF is combined with a synergistic cell attachment motif PHSRN, either through sequence engineering (Fmoc-FRGSFPHSRN) or through control over assembly properties (Fmoc-FRGDF/Fmoc-PHSRN coassembly). Here, the coassembled (Fmoc-FRGDF/Fmoc-PHSRN) system forms a synergistic network which promotes the attachment, proliferation and migration of muscle cells in vitro. The potential of Fmoc-SAP multi-sequence scaffolds is further investigated through the development of an artificial tumour microenvironment for cancer-cell studies. Here, Fmoc-FRGDF is combined with Fmoc-DIKVAV and used as a spheroid (LLC, NOR-10, LLC + NOR-10) micro-environment. The coassembled Fmoc-FRGDF/Fmoc-DIKVAV microenvironment enhances cancer-cell growth and progression compared to 2D cultures, non-encapsulate spheroids, and spheroids encapsulated in agarose. Agarose was selected as a control owing to the similar physical properties yet lack of biofunctionalisation. Results from this study reinforce the potential of Fmoc-SAPs as advanced microenvironments, and further support the ease of biological functionalisation inherent with this material. Further scaffold functionalisation is investigated through macromolecule addition. Here, one of two macromolecules are coassembled into a Fmoc-FRGDF network. The first macromolecule is fucoidan, a seaweed-derived polysaccharide with known anti-inflammatory properties, while the second is versican, a developmentally important proteoglycan which plays a variety of roles in muscle development. Versican was selected owing to its charge similarity to fucoidan, yet vastly different biological function. Fucoidan addition was found to increase fibre bundling and alter hydrogel mechanical properties, while versican addition had no substantial effect on hydrogel mechanics when compared to an Fmoc-FRGDF empty-vector control. Cell morphology was substantially altered by macromolecule addition, with fucoidan samples resulting in smaller, rounder cells with fewer multinucleated syncytia compared to an Fmoc-FRGDF control, while versican hydrogels showed an initial decrease in cell-size and multinucleation after 24h and a comparable cell-size and multinucleation following 72h. Here, it is possible that macromolecule addition perturbs cells attachment, and therefore, macromolecule selection is a key consideration. Interestingly, the regain of cell morphological characteristics in versican-containing hydrogels following 72h indicates the ability of cells to break-down versican, while the maintenance of small, round cells in the fucoidan hydrogels shows an inability for cells to break down fucoidan. The ability of Fmoc-SAPs to form components in bioinks is investigated through assembly with gelatin methacryloyl (GelMA) macromolecules. Initially, GelMA nanostructure and mechanical properties are investigated in response to increased degree of methacrylation or increased control. Here, structure-function relationships are drawn, and 18% methacryloyl Gelma (LM-GelMA) is selected for further bioink development owing to favourable thermoresponsive viscoelastic properties and improved strain tolerance. LM-GelMA assembly with coassembled Fmoc-FRGDF/Fmoc-PHSRN is investigated as a potential avenue to develop biologically and mechanically tuneable hydrogels. The incorporation of Fmoc-SAPs allows for control over sequence selection, while control over mechanical properties is offered through GelMA inclusion. LM-GelMA/Fmoc-FRGDF/Fmoc-PHSRN (FPG-Hybrid) bioinks demonstrate enhanced printability and are shown to support primary myoblast differentiation. The potential of Fmoc-SAP/GelMA bioinks to act as a modular bioink toolkit is further investigated through Fmoc-FRGDF/Fmoc-PHSRN substitution with Fmoc-DIKVAV, to develop a neural-suitable bioink (DIKVAV-Hybrid). This DIKVAV-Hybrid bioink demonstrated unique mechanical morphological properties and is shown to support rat cortical neurosphere viability. Throughout this project, the networks have been vigorously characterised through various analytical techniques, including micro/nanoimaging (Transmission electron microscopy, Atomic force microscopy, Cryo-scanning electron microscopy), Small-angle X-ray scattering, Small-angle neutron scattering, rheology, and spectroscopy; while the overall effectiveness of these systems have been analysed through in vitro muscle and neural cultures. Work detailed through this thesis aims to vigorously characterise Fmoc-SAP hydrogels and bioinks, providing the foundations for further biological studies and material optimisation

Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold

[EN] Ionic substitutions are a promising strategy to enhance the biological performance of calcium phosphates (CaP) and composite materials for bone tissue engineering applications. However, systematic studies have not been performed on multi-substituted organic/inorganic scaffolds. In this work, highly porous composite scaffolds based on CaPs substituted with Sr2+, Mg2+, Zn2+ and SeO3 2¿ ions, and chitosan have been prepared by freezegelation technique. The scaffolds have shown highly porous structure, with very well interconnected pores and homogeneously dispersed CaPs, and high stability during 28 days in the degradation medium. Osteogenic potential of human mesenchymal stem cells seeded on scaffolds has been determined by histological, immunohistochemical and RT-qPCR analysis of cultured cells in static and dynamic conditions. Results indicated that ionic substitutions have a beneficial effect on cells and tissues. The scaffolds with multi-substituted CaPs have shown increased expression of osteogenesis related markers and increased phosphate deposits, compared to the scaffolds with non-substituted CaPs.The financial supports of the European Regional Development Fund (grant KK.01.1.1.07.0014) , the PID2019-106000RB-C21/AEI/10.13039/501100011033 project from the Spanish Research Agency, and the L'Oreal-UNESCO "For Women in Science" Foundation are gratefully acknowledged.Ressler, A.; Antunovic, M.; Teruel Biosca, L.; Ferrer GG; Babic, S.; Urlic, I.; Ivankovic, M.... (2022). Osteogenic differentiation of human mesenchymal stem cells on substituted calcium phosphate/chitosan composite scaffold. Carbohydrate Polymers. 277:1-16. https://doi.org/10.1016/j.carbpol.2021.11888311627

Biohybrids for Neural Tracts Regeneration

[ES] Las lesiones del sistema nervioso que implican la interrupción de haces axonales son devastadoras para el individuo. La regeneración autónoma de los tractos axonales dañados o degenerados es poco frecuente, ya que intervienen una gran cantidad de factores que limitan esta recuperación. Hoy en día, la medicina convencional no cuenta con tratamientos efectivos y exitosos para estas lesiones, y el tratamiento de los síntomas suele ser la mejor solución. Para revertirlo y lograr la reconexión funcional de las neuronas, la ingeniería de tejidos actualmente opta por el uso de soportes tridimensionales biocompatibles, células y moléculas bioactivas. Específicamente, una de las estrategias propuestas han sido los conductos nerviosos guiados, no solo para lesiones de nervios periféricos sino también para tractos del sistema nervioso central. En esta Tesis Doctoral, se propone la combinación de un conducto tubular hueco de ácido hialurónico (HA) relleno con fibras de ácido poli-L-lactida (PLA) en su lumen, y con células de Schwann (SC) pre-cultivadas como células de soporte de la extension axonal para superar los obstáculos que limitan la regeneración de axones in vivo. Se ha demostrado que el conducto de HA y las fibras de PLA mantienen la proliferación de las SC, las cuales forman una estructura cilíndica denominada 'vaina de SC' en la pared interna del lumen del conducto y a su vez crecen de forma direccional en las fibras de PLA. El conjunto unidireccional paralelo formado por las fibras PLA y las SC recapitula las características direccionales de los tractos axonales en el sistema nervioso. Al sembrar un explante de ganglio de la raíz dorsal (DRG) en uno de los extremos del conducto, se ha conseguido el crecimiento de los axones del DRG y se ha estudiado las características de las SC, los axones crecidos y su asociación, comprobando que el biohíbrido es capaz de soportar el crecimiento axonal. Además, se propone un concepto multimodular para superar las limitaciones típicas de la regeneración axonal a larga distancia, con la combinación de haces de fibras de PLA en el lumen de varios conductos o módulos de HA individuales más cortos que se posicionan uno detrás del otro, diseñando conductos nerviosos guiados con la longitud deseada, junto con SC pre-cultivadas. El conducto multimodular demostró ser eficaz para promover el crecimiento dirigido de axones. Además, se ha desarrollado un constructo compuesto por la estructura formada por las fibras de PLA y las SC, denominado 'cordón neural', tras eliminar el conducto de HA, lo que abre la puerta a la generación de una estructura neural in vitro para su trasplante.[CA] Les lesions de el sistema nerviós que impliquen la interrupció de feixos axonals són devastadores per a l'individu. La regeneració autònoma dels tractes axonals danyats o degenerats és poc freqüent, ja que intervenen una gran quantitat de factors que limiten aquesta recuperació. Avui dia, la medicina convencional no compta amb tractaments efectius i reeixits per aquestes lesions, i el tractament dels símptomes sol ser la millor solució. Per revertir i aconseguir la reconnexió funcional de les neurones, l'enginyeria de teixits actualment opta per l'ús de suports tridimensionals biocompatibles, cèl·lules i molècules bioactives. Específicament, una de les estratègies proposades han estat els conductes nerviosos guiats, no només per lesions de nervis perifèrics sinó també per tractes de sistema nerviós central. En aquesta tesi doctoral, es proposa la combinació d'un conducte tubular buit d'àcid hialurònic (HA) farcit amb fibres d'àcid poli-L-lactida (PLA) en el seu lumen, i amb cèl·lules de Schwann (SC) pre-cultivades com a cèl·lules de suport de l'extension axonal per superar els obstacles que limiten la regeneració d'axons in vivo. S'ha demostrat que el conducte d'HA i les fibres de PLA mantenen la proliferació de les SC, les quals formen una estructura cilíndica anomenada 'beina de SC' a la paret interna de l'lumen de l'conducte i al seu torn creixen de manera direccional en les fibres de PLA. El conjunt unidireccional paral·lel format per les fibres PLA i les SC recapitula les característiques direccionals dels tractes axonals en el sistema nerviós. A l'sembrar un explantament de gangli de l'arrel dorsal (DRG) en un dels extrems de l'conducte, s'ha seguit el creixement dels axons de l'DRG i s'ha estudiat les característiques de les SC, els axons crescuts i la seva associació, comprovant que el biohíbrido és capaç de suportar el creixement axonal. A més, es proposa un concepte multimodular per superar les limitacions típiques de la regeneració axonal a llarga distància, amb la combinació de feixos de fibres de PLA en el lumen de diversos conductes o mòduls de HA individuals més curts que es posicionen un darrere l'l'altre, dissenyant conductes nerviosos guiats amb la longitud desitjada, juntament amb SC pre-cultivades. El conducte multimodular va demostrar ser eficaç per promoure el creixement dirigit d'axons. A més, s'ha desenvolupat un constructe format per l'estructura formada per les fibres de PLA i les SC, denominat 'cordó neural', després d'eliminar el conducte d'HA, el que obre la porta a la generació d'una estructura neural in vitro per al seu trasplantament.[EN] Injuries to the nervous system that involve the disruption of axonal bundles are devastating to the individual. Autonomous regeneration of damaged or degenerated axonal tracts is infrequent since a large number of factors are involved limiting this recovery. Nowadays, conventional medicine does not have effective and successful treatments for these injuries, and the treatment of symptoms is often the best solution. In order to reverse it and achieve the functional reconnection of neurons, tissue engineering currently opts for the use of biocompatible three-dimensional supports, cells, and bioactive molecules. Specifically, one of the proposed strategies has been nerve guidance conduits, not only for peripheral nerve injuries but also for tracts of the central nervous system. In this Doctoral Thesis, we propose the combination of hyaluronic acid (HA) single-channel tubular conduit filled with poly-L-lactide acid (PLA) fibres in its lumen, with pre-cultured Schwann cells (SC) as cells supportive of axon extension to overcome the obstacles limiting axon regeneration in vivo. We have proved that HA conduit and PLA fibres sustain the proliferation of SC, which form a cylindrical structure named 'SC sheath' on the inner wall of the lumen of the conduit and in turn grow directionally in the PLA fibres. The parallel unidirectional ensemble formed by PLA fibres and SC recapitulates the directional features of axonal pathways in the nervous system. Planting a dorsal root ganglion (DRG) explant on one of the conduit's ends, we have followed axon outgrowth from the DRG and studied the features of SC, the grown axons and their association, checking that the biohybrid is capable of supporting axonal growth. Furthermore, we propose a multimodular concept to overcome the typical limitations of long-distance axonal regeneration, with the combination of PLA fibres bundle in the lumen of several shorter individual HA conduits or modules which positioned themselves one behind the other, designing nerve guided conduits with the desired length, together with pre-cultured SC. The multimodular conduit proved effective in promoting directed axon growth. Moreover, we developed a construct consisting of the structure formed by the PLA fibres and the SC, named 'neural cord', after eliminating the HA conduit, that opens the door to the generation of a neural structure in vitro for transplantation.La presente tesis doctoral se ha realizado con la financiación del Ministerio de Economía y Competitividad a través de los proyectos MAT2015-66666-C3-1-R, DPI2015-72863-EXP, y AEI RTI2018-095872-B-C21-C22/ERDF. Agradezco también la beca FPU15/04975 al Ministerio de Educación Cultura y Deportes.Rodríguez Doblado, L. (2021). Biohybrids for Neural Tracts Regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16519