Dissertations / Theses on the topic 'Tubular regeneration'

ABSTRACT Peripheral nerve injury is a common clinical problem significantly affecting the patients’ quality of life. In case of severe transections, the bridging of the gap between the proximal and distal nerve stumps is required and autologous nerve grafts using sensory nerves (i.e. the sural nerve or antebrachial cutaneous nerve) are the current criterion standard. Nevertheless, donor-site morbidities, permanent loss of function, size mismatch between the donor nerve and the injured nerve and poor functional recovery rates have prompted the interest towards the identification of an alternative to this technique. To date, surgeons and researchers are turning their attention towards different grafts made of biological or artificial polymers. In fact, the development of hollow nerve guide conduits a) creating an adequate microenvironment for nutritional support/axons regeneration; b) acting as a barrier against the surrounding tissue infiltration; c) matching the effectiveness of the autologous nerve graft, would be beneficial to the field of peripheral nerve surgery. Over the years, many biomaterials of natural or synthetic origin and with different characteristics in terms of biodegradability have been studied. However, it has not been identified yet a prosthesis able to guarantee a better regenerated tissue than the others.
The aim of the present study was to manufacture and investigate in vitro and in vivo the characteristics and the regenerative potential of three different nerve conduits made up of polyvinyl alcohol (PVA); 1% Oxidized PVA (1% Ox PVA) and Silk-Fibroin (SF). While the use of PVA and SF for the realization of neuro-guides has already been studied in the past, oxidized PVA (recently patented by our research group) is a new material for this purpose. In parallel, this study also allowed to assess the quality of axonal regeneration guaranteed by neuro-guides with different origin (synthetic vs. natural) and biodegradation properties (vs non-biodegradable biodegradable). After preparing the three different polymer solutions, disk-shaped and tubular supports were manufactured. These were employed for in vitro and in vivo studies respectively. Considering in vitro analysis, a morphological characterization of supports was performed by Scanning Electron Microscopy (SEM). Thereafter, the biocompatibility and the biological activity of the three different scaffolds was assessed using a Schwann-cell line (SH-SY5Y). Cells were seeded on supports and their adhesion and proliferation was evaluated by SEM and MTT assay at two different end-points (3 and 7 days from seeding). Regarding in vivo tests, nerve conduits were implanted in animal models (Sprague-Dawley rats) of peripheral nerve injury with loss of substance (nerve gap: 5 mm). At 12-weeks from surgery, the functional recovery of the sciatic nerve was assessed; thereafter, the animals were euthanized and the dissection occurred. Prior to explant, the gross appearance of grafts was carefully observed in situ. Specimens were than processed for histological (hematoxylin and eosin staining) and immunohistochemical analysis (anti-CD3; anti-S100) as well as for further Transmission Electron Microscopy (TEM) analysis. The objective was to assess the quality of the regenerated nerve-tissue highlighting any differences in efficacy between the three types of nerve-conduits; to this end, the histomorphological analysis has been fundamental allowing to quantify the axons (myelinated vs unmyelinated nerve fibers) at different levels of the explant (proximal vs central vs distal portion); the controlateral sciatic nerve was used as control. Considering the in vitro results, SEM micrographs showed that PVA and SF supports have a smooth and regular surface; conversely, a certain roughness was noticed observing the ultrastructure of 1% Ox PVA disk-shaped scaffolds. Despite the superficial appearance of supports, it does not seem to affect the interaction with the cells. In fact, PVA-based scaffolds do not support cell adhesion and proliferation; SEM analysis and the MTT assay do not identified the presence of SH-SY5Y cells after 3 and 7 days from seeding. This result can be attributed to the high hydrophilic nature of the hydrogels. Conversely, SF scaffolds are adequate to promote SH-SY5Y cells growth. Regarding the in vivo study, all nerve conduits showed good characteristics in terms of handiness, being easy-suturing and demonstrating also an adequate tear-resistance feature; PVA-based scaffolds appear more flexible than SF guides. After 12 weeks from surgery, all animals showed a sciatic nerve functional recovery; in particular, all of them supported their body weight on the hind leg even though animals implanted with PVA and SF nerve conduits sometimes showed spasms during the walk while not limping. On the contrary, animals implanted with 1% Ox PVA nerve conduits exhibited a normal movement. At the time of dissection, the three scaffolds were still clearly identifiable. Any dislocation of the grafts or neuroma formation at the stumps was observed; moreover, the transparency of the three scaffolds allowed to identify the presence of a regenerated tissue inside. Thereafter, histological and immunohistochemical analysis were performed to evaluate the quality of axonal regeneration. Preliminarily, the haematoxylin and eosin staining of the specimens (cross-section of the central portion) highlighted the morphological integrity of the structure. In fact, three layers are recognizable proceeding from the periphery to the inside of the sections: an external fibrous capsule; a layer corresponding to the nerve conduit; a homogeneous and dense regenerated tissue in the middle. The biocompatibility of the grafts was verified by immunohistochemical analysis; anti-CD3 immunohistochemistry demonstrated the absence of severe inflammatory reactions. At the same time, several S100+ cells were identified suggesting the extensive presence of Schwann-cells. In parallel, the typical peripheral nerve morphology was highlighted also by Toluidine Blue staining by means of was considered also the appearance of the proximal and distal stumps. Although all samples support the recovery of the lesion, some differences can be found between the three experimental groups; these results were confirmed also by TEM micrographs. The histomorphometric analysis of samples evaluated the total axons number per nerve and axon density (axons/μm2); for each graft were considered the proximal, the central and the distal section. The collected data showed that 1% Ox PVA conduits assure a better outcome in nerve regeneration than the non-biodegradable PVA grafts which among the three groups proved to be the ones with the lower outcomes.
The results of this study showed that all nerve conduits considered (PVA; 1% Ox PVA and SF) promote peripheral nerve regeneration in case of neurotmesis with loss of substance. Considering the quality of regenerates, better outcomes were observed analyzing the 1% Ox PVA explants compared to PVA and Silk-Fibroin ones.
RIASSUNTO Le lesioni nervose periferiche costituiscono un problema clinico piuttosto comune, il quale inficia in modo significativo la qualità della vita dei pazienti. In caso di lesioni gravi con perdita di sostanza, al fine di colmare il gap tra il moncone prossimale ed il distale, il gold- standard prevede l’impianto di innesti nervosi autologhi utilizzando nervi sensoriali (ad es., nervo surale o nervo cutaneo antibrachiale). Tuttavia, criticità quali la morbidità del sito donatore, la perdita in funzionalità, la mancata corrispondenza dimensionale tra il nervo donatore ed il nervo lesionato oltre ad uno scarso recupero funzionale hanno spinto l'interesse verso l'identificazione di un approccio alternativo. Allo stato dell’arte, chirurghi e ricercatori stanno volgendo la loro attenzione verso innesti polimerici diversi (grafts) di natura sia biologica che artificiale. Infatti, lo sviluppo di neuroguide capaci di: a) creare un microambiente ideale per la rigenerazione assonale; b) fornire una protezione dall'infiltrazione di tessuto circostante; c) possedere un’efficacia analoga a quella garantita dall’innesto nervoso autologo; costituirebbe un vantaggio significativo nell’ambito della chirurgia del nervo periferico. Nel corso degli anni, sono stati studiati molti biomateriali di origine sia naturale che sintetica aventi caratteristiche differenti in termini di biodegradabilità. Tuttavia, considerando la qualità del tessuto rigenerato, non è ancora stata individuata una protesi più performante rispetto alle altre. L’obiettivo di questo studio è stato quello di allestire e studiare, sia in vitro che in vivo, le caratteristiche ed il potenziale rigenerativo di tre diverse neuroguide rispettivamente costituite da: alcool polivinilico (PVA); PVA ossidato 1% (PVA Ox 1%) e Fibroina della Seta (FS). Mentre l’impiego di PVA e FS per la realizzazione di grafts è già stato investigato in passato, il PVA Ox 1% (recentemente brevettato dal nostro gruppo di ricerca) costituisce un nuovo materiale per questo scopo. In parallelo, questo studio ha anche consentito di confrontare la qualità della rigenerazione assonale sostenuta da neuroguide diverse sia per origine (sintetica vs naturale) che per proprietà biodegradative (biodegradabili vs nonbiodegradabili).
Dopo aver allestito le tre diverse soluzioni polimeriche, sono stati quindi preparati scaffolds sia discoidali che in forma di graft tubulare, utilizzati rispettivamente per i successivi studi in vitro e in vivo. Nell’ambito degli studi in vitro, è stata effettuata una caratterizzazione morfologica dei supporti mediante microscopia elettronica a scansione (SEM). Successivamente, la biocompatibilità e l'attività biologica dei tre differenti scaffolds è stata valutata utilizzando una linea di cellule di Schwann (SH-SY5Y). Le cellule sono state seminate sui supporti e la loro adesione e la proliferazione è stata valutata mediante saggio MTT oltre che SEM a due differenti end-point (3 e 7 giorni dalla semina). Per quanto riguarda lo studio in vivo, i graft tubulari sono stati impiantati in modelli animali (ratti Sprague- Dawley) di lesione nervosa periferica con perdita di sostanza (gap tra moncone prossimale e distale: 5 mm). A 12 settimane dalla chirurgia, è stato valutato il recupero funzionale del nervo sciatico; successivamente, gli animali sono stati sacrificati. Dopo dissezione, prima di procedere all’espianto, l'aspetto macroscopico degli innesti è stato attentamente osservato in situ. I campioni sono stati quindi prelevati e trattati per le successive analisi istologiche (ematossilina ed eosina) ed immunoistochimiche (anti-CD3; anti-S100) nonché per ulteriori analisi di microscopia elettronica a scansione (TEM). L'obiettivo è stato quello di valutare la qualità del tessuto rigenerato evidenziando eventuali differenze di efficacia tra i tre tipi di grafts; a tal fine, anche l'analisi istomorfologica si è rivelata fondamentale, permettendo essa di quantificare gli assoni (mielinici vs amielinici) in diverse porzioni del campione (porzione prossimale vs centrale vs distale). Il nervo sciatico controlaterale è stato usato come controllo. Considerando i risultati degli studi in vitro, le immagini al SEM hanno mostrato come i supporti in PVA e FS mostrino una superficie liscia e regolare; al contrario, una certa ruvidità è stata notata osservando l’ultrastruttura degli scaffold discoidali in PVA Ox 1%. Nonostante il diverso aspetto ultrastrutturale dei supporti, esso non sembra influenzare l'interazione con le cellule. Il PVA (sia nativo che ossidato) non sostiene l'adesione e la proliferazione cellulare; infatti, sia le analisi al SEM che il saggio MTT non hanno identificato la presenza di cellule SH-SY5Y dopo 3 e 7 giorni dalla semina. Questo risultato può essere attribuito alla elevata idrofilia degli idrogeli Al contrario, gli scaffold in FS sono adeguati per promuovere la crescita delle SH-SY5Y. Per quanto riguarda lo studio in vivo, tutti i graft mostrato buone caratteristiche in termini di manipolabilità, essendo facilmente suturabili e dimostrando anche una adeguata resistenza allo strappo; gli scaffold in PVA appaiono più flessibile rispetto alle guide in FS. Dopo 12 settimane dalla chirurgia, tutti gli animali hanno mostrato un certo recupero funzionale dell’arto operato; in particolare, tutti distribuivano il proprio peso corporeo anche sulla zampa posteriore. Pur non zoppicando, gli animali impiantati con PVA e SF mostravano talvolta degli spasmi durante la deambulazione, al contrario, gli animali impiantati con graft in PVA Ox 1% esibivano un movimento normale. Al momento della dissezione, i tre graft erano ancora chiaramente identificabili. Non è stata riscontrata alcuna dislocazione degli innesti o formazione di neuroma in corrispondenza dei monconi; inoltre, la trasparenza delle tre neuroguide ha permesso di identificare la presenza di un tessuto rigenerato al loro interno. Successivamente, sono state effettuate analisi istologiche ed immunoistochimiche per valutare la qualità della rigenerazione assonale. Preliminarmente, mediante colorazione con ematossilina ed eosina (sezione trasversale della porzione centrale) è stato possibile mettere in evidenza l'integrità morfologica della struttura. Procedendo dalla periferia della sezione verso l'interno sono riconoscibili: una capsula fibrosa esterna; il graft; ed il tessuto neo-rigenerato, omogeneo e denso, nel mezzo. La biocompatibilità degli innesti è stata verificata mediante analisi immunoistochimica; la scarsa presenza di cellule CD3+ ha dimostrato l'assenza di reazioni infiammatorie gravi riconducibili all’impianto. Contestualmente, l’elevata presenza di elementi S100+ riscontrata in tutti i campioni ha comprovato una evidente rigenerazione assonale. In parallelo, la morfologia tipica del tessuto nervoso periferico è stata altresì evidenziata mediante colorazione con Blu di Toluidina mediante la quale è stato considerato anche l'aspetto dei monconi prossimale e distale.
Sebbene tutti i campioni supportino il recupero della lesione, alcune differenze possono essere riscontrate tra i tre gruppi sperimentali; questi risultati sono stati confermati anche dalle micrografie al TEM. L'analisi morfometrica dei campioni ha valutato il numero totale di assoni/nervo e la loro densità (assoni / μm2); per ogni innesto sono state considerate le sezioni prossimale, centrale e distale. I dati raccolti hanno dimostrato che il PVA Ox 1% assicura un risultato migliore nella rigenerazione assonale rispetto agli innesti non biodegradabili in PVA, il quale tra i tre gruppi è risultato essere quello con l’outcome inferiore. I risultati di questo studio hanno mostrato che, in caso di neurotmesi con perdita di sostanza, tutti i graft allestiti (PVA; PVA Ox 1% e FS) promuovono la rigenerazione del nervo. Considerando la qualità del tessuto rigenerato, sono stati osservati dei risultati migliori con graft in PVA Ox 1% rispetto a quelli ottenuti da neuroguide in PVA e FS.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2000.
Includes bibliographical references (leaves 143-148).
Peripheral nerve injury affects nearly 200,000 patients annually in the United States and unless treated results in paralysis of skeletal muscle and loss of sensation. Previous studies in this laboratory have focused on comparing the effectiveness of various tubular devices in repairing experimental nerve injuries in an animal model. The devices were rank-ordered based on clinically relevant assays of regeneration such as number regenerated nerve fibers and electrophysiological conduction properties of the regenerated nerves. Such assays provide a useful measure of the clinical efficacy of devices but require long-term (up to 60-week) studies in order to obtain meaningful results. There exists a need for a short-term (less than 12-week) assay with which nerve repair devices can be compared. The overall goal of this thesis was to establish an experimental assay that can be used to detect statistically significant differences among nerve repair devices in short-term studies. In this thesis, four different assays of nerve regeneration were compared on the basis of their appropriateness to quantify the regeneration promoted by nerve repair devices in studies less than 12 weeks in duration. An acceptable assay must reach a plateau with time during short-term studies and must yield a quantitative metric with which nerve devices can be compared. The results of this thesis suggest that an assay based on ability of a nerve repair device to promote reinnervation across nerve gaps of various lengths meets the criteria for an acceptable assay. The data also indicate that the characteristic gap length (Lc), which is derived from curve-fitting the experimental data for reinnervation versus gap length, can be used as a quantitative metric of nerve regeneration. The experimental data indicate that for the silicone tube device, the value of Lc reached a plateau with time before 9 weeks, and the standard error in L was less than 5 percent of the value in two different nerve repair models (single-leg and crossanastomosis). The data also suggest that statistically significant differences between the silicone tube device and a collagen-based device (the CG device) are obtainable during short-term (12-week) studies.
by Mark H. Spilker.
Ph.D.

Orientador: Eliana Aparecida de Resende Duek
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica
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Mestrado
Materiais e Processos de Fabricação
Mestre em Engenharia Mecânica

During the central nervous system (CNS) morphogenesis, chemical gradients of morphogens such as retinoic acid (RA) or sonic hedgehog play a central role in regulating CNS patterning and differentiation of neural subtypes. Recapitulation of these gradients in a 3D hydrogel matrix may provide a model for CNS tissue formation in vitro. 3D Printing technology offers an opportunity to reproduce the complex architecture of cell microenvironment. We have developed a 3D-printable alginate hydrogel bioink suitable for extrusion-based bioprinting. The bioink was characterised by shear thinning, high printing resolution and minimal adverse effects on cell viability. The bioink was successfully used to print mouse embryonic stem cells (mESCs)-laden constructs and supported their differentiation into neural-like cells. Extrusion-based bioprinting was used to 3D-print hybrid polycaprolactone (PCL)-alginate tubular scaffolds functionalised with a fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) concentration gradient pattern. Quantification of the FITC-BSA concentrations in the scaffold showed a linear reduction in concentration as a function of scaffold’s distance (length). Tubular scaffolds printed with fibroblast-laden alginate supported cell viability and proliferation up to 6 days after printing. Next, the developed model was used to replicate the in vivo RA-induced directed differentiation of mESCs into spinal cord neurons. RA-concentration-dependent acquisition of neural identity was investigated using immunocytochemistry and flow cytometry analysis. RA promoted the formation of neurons with hindbrain and spinal cord identity and supressed the forebrain identity in a concentration-dependent manner. Among the investigated hydrogels, gelatine methacrylate (GelMA) supported neural differentiation and neurite outgrowth of the mESCs-derived embryoid bodies (EBs). Subsequently EBs-laden GelMA (5%) was successfully used as a bioink to print the hybrid PCL-hydrogel scaffolds. 3D Printing of EBs and RA-loaded GelMA in PCL scaffold induced differentiation of EBs into neurons with spinal cord positional identity. In conclusion, the model can be used for effective morphogens gradients delivery to replicate some of the complex processes of CNS development in vitro.

Objetivamos o estudo quantitativo e temporal do padrão de inervação do músculo extensor longo dos dedos (edl), após transecção do nervo ciático em camundongos e reparo imediato pela técnica de tubulização. Assim, o músculo edl apresentou-se desnervado na segunda semana após a tubulização. Todas as fibras musculares do edl estavam reinervados na sexta semana após o implante da protese tubular. Entre a sexta e a quadragesima semanas pós-implante a proporção de fibras musculares do edl com mono e poli-inervação aumentou de 3:1 para 4:1. Na quadragésima semana após o implante do tubo foi atingida a porcentagem máxima (80%) de inervação troncular das placas motoras do edl mono-inervadas. Mesmo com tempos prolongados de sobrevivencia após implante, não houve estabilização do padrão de origem das fibras nervosas que convergiam para as placas poli-inervadas do edl. A mono-inervação troncular foi readquirida por 65% das fibras musculares do edl dos animais tubulizados, padrão encontrado em 100% das fibras musculares do edl dos animais não operados. Os padrões morfometricos de reinervação do músculo edl foram idênticos, a partir do terceiro mes, entre o grupo de animais com secção e tubulização do nervo ciatico e o grupo com simples esmagamento do nervo, evidenciando padrão similar de reinervação muscular a longo prazo.
We aimed to study the quantitative and temporal pattern of muscle innervation extensor digitorum longus (EDL), following transection of the sciatic nerve in mice and immediate repair technique for tubing. Thus, the EDL muscle denervation presented in the second week after the tubing. All of the EDL muscle fibers were reinnervated in the sixth week after the implantation of prosthetic tube. Between the sixth and FORTY weeks post-implantation, the proportion of muscle fibers of the EDL with mono-and poly-innervation increased from 3:1 to 4:1. The forty weeks after the implantation of the tube was reached maximum percentage (80%) of trunk innervation of motor endplates of the EDL mono-innervated. Even with prolonged survival times after implantation, there was no stabilization of the pattern of origin of nerve fibers that converged on the plates of poly-innervated EDL. The mono-innervation trunk was regained by 65% of EDL muscle fibers of animals tubularized, pattern found in 100% of EDL muscle fibers of animals not operated. The morphometric patterns of reinnervation of the EDL muscle were identical, from the third month, the group of animals with tubing and resources section of the sciatic nerve and the group with simple nerve crush, showing similar pattern of muscle reinnervation in the long term.

Evaluation of tubular regenerative response in AKI-induced trasngenic rodent models. Discovered the existence of a progenitor population scattered in kidney tubule of adut mice devoted to tubular epithelium recovery after ischemic injury.

Indiana University-Purdue University Indianapolis (IUPUI)
Background: Root canal disinfection is an essential requirement for the success of regenerative endodontics. Currently, the so-called triple antibiotic paste (TAP) is considered the standard of care. Notwithstanding the good antimicrobial capacity, the high concentration of TAP has shown significant toxicity to human cells, especially dental pulp stem cells. A novel drug release system, i.e., a triple antibiotic paste-mimic electrospun scaffold containing low concentrations of the antibiotics present in the TAP, has emerged as an effective and reliable alternative to fight root canal infections without potential toxic effects on dental stem cells, which are an integral part of the regenerative treatment. Objectives: The aim of this study was to determine the antimicrobial efficacy of an innovative three-dimensional (3D) triple antibiotic paste-mimic tubular scaffold against Actinomyces naeslundii biofilm formed inside human root canal dentinal tubules. Materials and methods: Pure polydioxanone (PDS) polymer solution and PDS loaded with metronidazole, ciprofloxacin and minocycline (35 wt.% of each antibiotic, 3D-TAP-mimic scaffold) were spun into 3D fibrous scaffolds. A. naeslundii (ATCC 43146) was centrifuged to induce biofilm formation inside human root canal dentinal tubules using a dentin slice model (1 mm thickness and 2.5 mm canal diameter). The infected dentin slices were exposed to the 3D-TAP-mimic scaffold, TAP solution (50 mg/mL of each antibiotic), and antibiotic-free PDS. Biofilm elimination was quantitatively and qualitatively analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Results: A dense penetration of A. naeslundii biofilm was observed by CLSM throughout the dentinal tubules. 3D-TAP-mimic scaffold significantly reduced the percentage of viable bacteria compared with PDS (p <.05). TAP solution completely eliminated viable bacteria without differing from 3D-TAP-mimic scaffolds. SEM images showed results similar to CLSM. Conclusion: Collectively, the proposed tubular 3D-TAP-mimic scaffold holds significant clinical potential for root canal disinfection strategy prior to regenerative endodontics.

Esta dissertação centra-se no estudo da viabilidade energética de amortecedores regenerativos de relutância magnética em veículos elétricos. Neste trabalho procura-se contribuir para investigação e desenvolvimento de geradores lineares de relutância variável comutados de topologia tubular com aplicação em sistemas de suspensão regenerativa para conversão de energia de vibração. É descrito o sistema de suspensão, é definido o modelo matemático que rege o seu comportamento e os diferentes tipos de suspensão. Os veículos elétricos têm ganho muito interesse, como alternativa aos veículos movidos por combustíveis fosseis, por serem mais ecológicos. É feita a descrição da evolução dos veículos elétricos desde a primeira geração até à quarta geração. Entretanto, aborda-se o princípio de conversão de energia da máquina linear de relutância variável comutada, estuda-se o modelo dinâmico do gerador, e referem-se os conversores eletrónicos de potência para o seu controlo. Por fim, é feita a análise energética da suspensão regenerativa com o gerador linear de relutância variável comutado, e todo processo de cálculo computacional é implementado em linguagem própria do software Matlab e do software gratuito Finite Element Method Magnetics (FEMM).
This Dissertation focuses on the study of the energetic feasibility of magnetic switched reluctance regenerative dampers in electric vehicles. In this work, it is intended to contribute to research and development of linear switched reluctance generators with tubular topology for application in regenerative suspension systems for vibration energy conversion. The suspension system is described, the mathematical model that governs its behavior and the different type of suspensions are defined. Electric vehicles have gained a lot of interest, as an alternative to fossil-powered vehicles, because they are more ecological. Furthermore, the principle of energy conversion in linear switched reluctance machine is discussed, the dynamic model of the generator is studied, and the electronic power converters for its control are presented. Lastly, there is an energy analysis of the regenerative suspension based on linear switched reluctance generator. The computational calculation process is implemented using Matlab and Finite Element Method Magnetics (FEMM) software.