Tuneable hydrogel platform for oligonucleotide biomarkers detection

Early detection of circulating biomarkers in human fluids can improve the quality of life reducing the development of several deadliest diseases. Among the latest and most significant medical concept “Liquid Biopsy” is emerging as non-invasive method of gleaning insight into the dynamics of diseases through a patient fluid sample. Actually many tests have been developed in this context, however, despite all the efforts, the majority are complex, require extensive manipulations and skilled operators, failing for point-of-care (POC) applications. The main focus of this thesis has been devoted to develop advanced technologies, based on a hydrogel platform, properly designed for biosensing application. In particular, PEG engineered hydrogel microparticles have been synthetized with different chemical strategies and functionalized with oligonucleotide probes to detect circulating biomarker in human serum. In this thesis, the parameters affecting the hydrogel biosensing properties have been carefully evaluated to obtain an accurate functionalized network capable of sensitive and specific biomarker recognition. The developed hydrogel assays are based on the optical fluorescence read out over a single microgel, fixed the number of microgels and sample volume for each test. Therefore, the target concentration is easily quantified comparing the fluorescence observed with a calibration curve. The thesis starts with the description of a microgel-based bioassay for Cytomegalovirus infection diagnosis (Chapter 2). The bioassay is based on microgels, with core-shell architecture, endowed with optical fluorescence probes for the recognition of circulating endogenous viral hcmv-miR-US4-5p. Then, a microgel-based bioassay for microRNA biomarker detection in cancer application is reported (Chapter 3). In this case, the outmost shell of microgel is functionalized with molecular beacons for circulating miR-21 recognition. In Chapter 4 are elucidated the main parameters taken into consideration to develop sensitive and specific microgel-based assay for long oligonucleotide detection, as lncRNA, mRNA or DNA. In particular, core-shell microgels functionalized with double strand or molecular beacon probes are compared in terms of sensitivity, sensibility and assay time. Finally, is described the design of Three-dimensional hydrogel microparticles in microfluidics for the detection of microRNA and in particular is presented the case study of miR-143-3p detection as early biomarker in Amyotrophic lateral sclerosis (Chapter 5). Results show that finely tuning both the probe density and the number of microparticles per assay are achieved appealing limits of detection, avoiding amplification steps. The molecular beacon-microgels assay further reduces the characteristics time of hybridization observed in beads based assay and is extremely specific towards single mutated targets (SNP). Due to the PEG anti-fouling property, target detection occurs in human serum without loss in sensitivity. Moreover, the hydrogel-based assays are suitable for several laboratory equipment, are stable over a one-year span time and work in low sample volume. In addition to the mentioned advantages, microfluidics approach significantly reduces costs and the time of production resulting attractive for industrial production. Therefore, the biosensing platform obtained using engineered hydrogels can represent a smart technology capable to predict, identify and follow-up several diseases, monitoring free circulating oligonucleotides in body fluids. The flexible use of these engineered hydrogels, which avoid sample manipulation and can be easily integrated into miniaturized device for optical readout, aims to push these technologies as point-of-care device

Bioengineered Scaffolds for Peripheral Nerve Regeneration

Nerve autografts are widely used clinically to repair nerve grafts. However, nerve grafts have many limitations, such as, availability of donor nerve grafts, and loss of function at donor site. To overcome these problems, we have used a tissue engineering approach to design three-dimensional (3D) agarose scaffolds containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) to mimic in vivo conditions to promote nerve regeneration in rats. To determine the effect of LN-1 gradients on neurite extension in vitro, dorsal root ganglia (DRG) from chick embryos were cultured in 3D hydrogels. A gradient of LN-1 molecules in agarose gels was made by diffusion technique. LN-1 was then immobilized to the agarose hydrogels using a photo-crosslinker, Sulfo-SANPAH (Sulfosuccinimidyl-6-[4-azido-2-nitrophenylamino] hexanoate). Anisotropic scaffolds with three different slopes of LN-1 gradients were used. Isotropic scaffolds with uniform concentrations of LN-1, at various levels, were used as a positive control. DRG cultured in anisotropic scaffolds with optimal slope of LN-1 gradient extended neurites twice as fast as DRG in optimal concentration in isotropic scaffolds. Also, in the anisotropic scaffolds the faster growing neurites were aligned along the direction of LN-1 gradient. To promote nerve regeneration in vivo, tubular polysulfone guidance channels containing agarose hydrogels with gradients of LN-1 and NGF (anisotropic scaffolds) were used to bridge 20-mm nerve gaps in rats. Nerve autografts were used as positive controls and isotropic scaffolds, with uniform concentration of LN-1 and NGF, were used as negative controls. After 4-months, the rats were sacrificed and nerve histology was done to test for nerve regeneration. Only anisotropic scaffolds and nerve autografts contained evidence of axonal regeneration. Both groups had similar numbers of myelinated axons and similar axonal-diameter distribution. However, nerve graft group performed better in functional outcome as measured by relative gastrocnemius muscle weight (RGMW) and electrophysiology. Optimization of performance of anisotropic scaffolds by varying the LN-1 and NGF concentration gradients might lead to development of scaffolds that can perform as well as nerve auotgrafts for nerve regeneration over long nerve gaps.Ph.D.Committee Chair: Bellamkonda, Ravi; Committee Member: English, Arthur; Committee Member: Garcia, Andres; Committee Member: LaPlaca, Michelle; Committee Member: McDevitt, Tod

Carboxymethyl cellulose-based cryogels as scaffolds for pancreatic and skeletal muscle tissue engineering

[eng] Diabetes incidence highly increased in the last years. According to IDF (International Diabetes Federation), 463 million people suffered this disease in 2019. The estimations of diabetic people highly increase in the upcoming years, rising approximately to 700 million diabetic patients in 2045 [1]. Type 2 diabetes (T2D) is the most common type of diabetes, representing 90% of diabetic patients. It occurs when the body becomes resistant to insulin. Body insulin resistance confirms that T2D is not only a pancreatic disease, as there are many other tissues involved, like liver, adipose tissue, or skeletal muscle. This last has a significant implication in glucose-insulin homeostasis as it is one of the main glucose-consuming organs in the body. Nowadays, to study how two tissues crosstalk between them, animal testing is the gold standard. However, the unmatching physiological behaviors compared to humans, the variability between different animals, ethical dilemmas, and the need to go for more personalized medicine activates the search for other suitable alternatives. At this point, Organs-on-a-chip appeared as a valid alternative. Organs-on-a-chip (OOC) are 3D bioengineered microfluidic cell culture platforms to simulate microphysological environments of an organ or its specific functions. Nowadays, to engineer the tissues for OOC applications, encapsulating cells inside hydrogels is the most common technique. Its beneficial properties include high water content, mechanical adjustability, and moldability to generate the desired architectures [2]. However, its small porosity limits nutrient and oxygen diffusion through it [3]. This problem is a significant limitation when pancreatic islets are encapsulated inside hydrogels due to their size (~100 μm of diameter). Pancreatic islets are cell aggregations composed of many different cells as insulin-secreting cells (Beta-cells) or glucagon-secreting cells (alpha-cells). Similarly, skeletal muscle tissue is generally encapsulated in small bundles. Skeletal muscle is a highly aligned and multinucleated tissue formed from the fusion of single cells, called myoblasts, into multinucleated cells, called myotubes. Cryogels have been proposed as a valid alternative to overcome these limitations. Cryogels are fabricated by crosslinking a prepolymer solution at sub-zero temperatures, so while the material crosslinks, water freezes, generating the desired micropore architecture. After thawing, cryogels are sponge-like scaffolds with microporous structure, high interconnected porosity, high diffusivity, fine-tuned properties, and desired internal pore architecture. This thesis developed two cryogel scaffolds made of gelatin and carboxymethylcellulose with different pore architectures to engineer pancreatic and skeletal muscle tissues. Here, we proved that the achieved pore architecture fits with the prerequisites to engineer each tissue. Moreover, the mechanical and physical properties of each scaffold highly resemble the 3D microenvironment of each tissue. In pancreatic tissue, we generate a random pore cryogel to aggregate beta-cells to form pseudoislets. We proved that these engineered pseudoislets are viable, functional responding correctly to the glucose and improving insulin response compared to monolayer results. In the skeletal muscle approach, we could develop a highly aligned pore architecture to prompt cell alignment and cell fusion. Moreover, we incorporate carbon nanotubes to enhance the electrical conductivity of the scaffold, so by applying electrical pulse stimulation, we could improve the early steps of the myogenic maturation.[cat] La incidència de la diabetis ha augmentat considerablement en els últims anys. Segons l’IDF (International Diabetes Federation), al 2019 hi havia 463 milions de persones que patien diabetis i les estimacions estimen un augment considerable de casos, arribant als 700 milions de persones diabètiques cap al 2045 [1]. Entre els diferents tipus de diabetis, la diabetis tipus 2, és la que té major incidència en la població, corresponent al 90% dels casos de pacients amb diabetis. Aquest tipus de diabetis, succeeix quan el cos es torna resistent a la insulina. Aquesta resistència a la insulina per part dels teixits perifèrics ens prova que la diabetis no és només una malaltia del pàncreas, sinó que hi ha altres teixits relacionats, com el fetge, el teixit adipós o el múscul esquelètic. Aquest últim té un factor molt rellevant en la homeòstasi de la insulina i la glucosa, ja que és un dels principals teixits consumidors de glucosa. La interacció, però, entre aquest dos teixits encara presenta molts interrogants. Actualment, per estudiar com dos teixits interactuen entre ells, el testeig animal és el mètode més confiable. No obstant, presenta certes limitacions, com la poca similitud en quan a l’activitat dels illots, la variabilitat fisiològica entre diferents animals, dilemes ètics o la necessitat d’encarar la recerca cap a una medicina més personalitzada. Aquesta finalitat és el que ha portat als científics a buscar alternatives a l’experimentació animal. Entre moltes, una de les més prometedores són els anomenats Òrgans-en-un-xip, plataformes 3D de cultiu cel·lular combinades amb microfluídica i biomaterials que permeten simular les funcions específiques d’un òrgan. Per tal de generar el teixit dins d’aquesta plataforma, l’encapsulació de cèl·lules dins de hidrogels és la tècnica més utilitzada, degut al seu alt contingut d’aigua, la seva adaptabilitat mecànica o la possibilitat de generar una certa estructura geomètrica [2]. No obstant, la seva petita porositat, limita la difusió homogènia d’oxigen i de nutrients dins seu [3]. Aquest problema creix quan es volen encapsular illots pancreàtics en bastides d’hidrogel, degut a la seva mida (~100 μm de diàmetre). Els illots pancreàtics són agregacions de varis tipus diferent de cèl·lules, on destaquen les cèl·lules secretores de insulina (cèl·lules beta) i les secretores de glucagó (cèl·lules alfa). Per altre costat, el teixit muscular s’encapsula en petits constructes per tal d’imitar l’estructura d’aquest. El múscul esquelètic és un teixit altament alineat, amb cèl·lules multi nucleades, anomenades miotubs, que s’obtenen a partir de la fusió de cèl·lules soles, anomenades mioblasts. Per tal de solucionar aquests problemes, els criogels han aparegut com a alternativa. Els criogels, estan fabricats a temperatures sota zero, així mentres el polímer crosslinca es formen cristalls de gel. Un cop formada la matriu, la bastida es descongela i aquests cristalls es desfaran, deixant pas a espais buits, anomenats pors. Aquests, seran els que posteriorment li donaran la l’estructura porosa, altament interconnectada, amb alta permeabilitat i amb una arquitectura de pors determinada a la nostra bestida. En aquesta tesi s’han desenvolupat dos bastides de cel·lulosa carboxymetilada diferents seguint la tècnica de la criogelificació. Cada bastida ha estat dissenyada per tenir una distribució i una arquitectura de pors diferent d’acord amb la necessitat i propietat del teixit que es vulgui generar. A més, les propietats físiques i mecàniques de les dos bastides tenen alta semblança amb les propietats físiques i mecàniques de la matriu extracel·lular de cada teixit. Per el teixit pancreàtic, s’ha generat una bastida amb un diàmetre de pors similar als illots pancreàtics, per tal que, sembrant cèl·lules beta, aquestes formin pseudoillots similars als illots fisiològics. A més, s’ha demostrat que aquests illots tenen el diàmetre i la arquitectura desitjada, són viables i capaços de respondre a diferents nivells de glucosa. A més, s’ha demostrat que aquestes cèl·lules agregades en forma de pseudoillots responen millor a la glucosa que les cèl·lules configurades en distribució dispersa. En el cas del múscul esquelètic, s’ha desenvolupat una bastida amb una arquitectura de pors altament alineada per promoure l’alineament cel·lular i la fusió cel·lular. A més, s’han pogut incorporar nanotubs de carboni per millorar les propietats elèctriques de la vestida. D’aquesta manera, aplicant pulsos elèctrics per estimular el teixit, s’han pogut millorar les etapes primerenques de la maduració miogènica

Toward personalized medicine: human adipose-derived stem cells (ASCs) and xenogenic-free media as enabling tools in tendon tissue engineering strategies.

L'abstract è presente nell'allegato / the abstract is in the attachmen

Design and development of an implantable biohybrid device for muscle stimulation following lower motor neuron injury

In the absence of innervation caused by complete lower motor neuron injuries, skeletal muscle undergoes an inexorable course of degeneration and atrophy. The most apparent and debilitating clinical outcome of denervation is the immediate loss of voluntary use of muscle. However, these injuries are associated with secondary complications of bones, skin and cardiovascular system that, if untreated, may be fatal. Electrical stimulation has been implemented as a clinical rehabilitation technique in patients with denervated degenerated muscles offering remarkable improvements in muscle function. Nevertheless, this approach has limitations and side effects triggered by the delivery of high intensity electrical pulses. Combining innovative approaches in the fields of cell therapy and implanted electronics offers the opportunity to develop a biohybrid device to stimulate muscles in patients with lower motor neuron injuries. Incorporation of stem cell-derived motor neurons into implantable electrodes, could allow muscles to be stimulated in a physiological manner and circumvent problems associated with direct stimulation of muscle. The hypothesis underpinning this project is that artificially-grown motor neurons can serve as an intermediate between stimulator and muscle, converting the electrical stimulus into a biological action potential and re-innervating muscle via neuromuscular interaction. Here, a suitable stem cell candidate with therapeutic potential was identified and a differentiation protocol developed to generate motor neuron-like cells. Thick-film technology and laser micromachining were implemented to manufacture electrode arrays with features and dimensions suitable for implantation. Manufactured electrodes were electrochemically characterised, and motor neuron-like cells incorporated to create biohybrid devices. In vitro results indicate manufactured electrodes support motor neuron-like cell growth and neurite extension. Moreover, electrochemical characterisation suggests electrodes are suitable for stimulation. Preliminary in vivo testing explored implantation in a rat muscle denervation model. Overall, this thesis demonstrates initial development of a novel approach for fabricating biohybrid devices that may improve stimulation of denervated muscles

Development of Tethered Aligned Engineered Neural Tissue Containing Elongated Neurons for Peripheral Nerve Regeneration

Following peripheral nerve injury, the axons in the distal nerve between the injury site and the muscle degenerate. When the injured site is very proximal, functional recovery from nerve repair is a clinical challenge since neuronal regeneration rate is limited, resulting in muscle atrophy due to the delay in reinnervation, even where the ‘gold standard’ autograft is used. Much research focuses on developing biomaterial scaffolds that mimic the autograft and promote host neurite regeneration from proximal to distal stump, whereas here, we aim to improve long distance repair by populating constructs with functional neurons and glial cells. With an engineered living scaffold populated with neurons exhibiting long neurite extensions supported by glial cells, the gap between proximal stump and muscle could potentially be reconnected promptly once the challenge of integration is overcome. To test the concept, a method was developed using tethered aligned engineered neural tissue (TaeNT) formed from simultaneous self-alignment of Schwann cells and collagen fibrils in a fully-hydrated tethered gel resulting in an anisotropic tissue-like structure. The in vitro results showed neurite elongation and alignment in the co-culture of neurons and Schwann cells in TaeNT, indicating that TaeNT could be an appropriate substrate for growing long neurites with a view to generating therapeutic constructs containing long functional neurons. The implantation of TaeNT containing neurons and Schwann cells in a 10mm-gap rat sciatic nerve for 3 weeks provided information about host-transplant cell interaction including Schwann cell migration and alignment inside the conduit, and neurite elongation across the conduit interface. Furthermore, in an attempt to induce longer neurite growth, TaeNT was proposed as a substrate that could be combined with mechanical tension application using a 3D-printed mould developed to stretch the cellular gels in a controlled manner. A series of newly designed protocols for mechanical tension application to induce growth response for enhanced neural regeneration was developed and discussed correspondingly. In summary, the findings represent the development and investigation of the regenerative potential for engineered living scaffolds containing neurons and Schwann cells suitable for stretch-growth to provide an elongated functional nerve graft. With a view to translation for clinical use, investigating the source of therapeutic cells in the conduit and the functional integration of host and transplanted cells is an important step towards optimising the regenerative potential of the engineered living scaffold

Shifting the Balance of Inflammatory and Pro-Resolving Lipid Mediators in Volumetric Muscle Loss Injuries

Extremity trauma involving large tissue loss (e.g., VML) presents a significant clinical challenge for both general and military populations. The frank loss of musculature characteristic of VML sufficiently disrupts or eliminates the wound’s endogenous repair mechanisms; thus, healing becomes difficult and often results in substantial scar tissue formation, permanent functional impairments, and chronic pain. Skeletal muscle is not well adapted to chronic injury stimulus and host mechanisms of inflammation resolution fail to trigger the shift in local immune cells from a transient pro-inflammatory state to a pro-resolving and pro-regenerative state. To explore the potential molecular facilitators of this persistent inflammation, we examined the biosynthesis of inflammatory lipid mediators and specialized pro-resolving lipid mediators (SPM) after VML injury. Our analysis suggests that critical size VML defects are unable to biosynthesize SPMs after injury and are, thus, burdened by a dysregulated and persistent inflammation. Therefore, we leveraged a modular polyethylene glycol-maleimide biomaterial platform to enable local release of a stable isomer of Resolvin D1 (AT-RvD1) in order to promote endogenous pathways of inflammation resolution. We show that the local delivery of AT-RvD1 is able to promote the molecular and cellular resolution of inflammation, enhance muscle regeneration and significantly improve muscle function after VML. These findings represent an improved understanding of the role of SPMs in the pathogenesis of VML and establish the pro-resolving hydrogel therapeutic as a way to promote functional muscle regeneration after traumatic injury.Ph.D

Mechanoresponsive drug delivery materials

Stimuli-responsive drug delivery materials release their payloads in response to physiological or external cues and are widely reported for stimuli such as pH, temperature, ionic strength, electrical potential, or applied magnetic field. While a handful of reports exist on materials responsive to mechanical stimuli, this area receives considerably less attention. This dissertation therefore explores three-dimensional networks and polymer-metal composites as mechanoresponsive biomaterials by using mechanical force to either trigger the release of entrapped agents or change the conformation of implants. At the nanoscale, shear is demonstrated as a mechanical stimulus for the release of a monoclonal antibody from nanofibrous, low molecular weight hydrogels formed from bio-inspired small molecule gelators. Using their self-healing, shear-thinning properties, mechanoresponsive neutralization of tumor necrosis factor alpha (TNFα) in a cell culture bioassay is achieved, suggesting utility for treating rheumatoid arthritis. Reaching the microscale, mechanical considerations are incorporated within the design of cisplatin-loaded meshes for sustained local drug delivery, which are fabricated through electrospinning a blend of polycaprolactone and poly(caprolactone-co-glycerol monostearate). These meshes are compliant, amenable to stapling/suturing, and they exhibit bulk superhydrophobicity (i.e., extraordinary resistance to wetting), which sustains release of cisplatin >90 days in vitro and significantly delays tumor recurrence in an in vivo murine lung cancer resection model. This polymer chemistry/processing strategy is then generalized by applying it to the poly(lactide-co-glycolide) family of biomedical polymers. As a macroscopic approach, a tunable, tension-responsive multilayered drug delivery device is developed, which consists of a water-absorbent core flanked by two superhydrophobic microparticle coatings. Applied strain initiates coating fracture to cause core hydration and subsequent drug release, with rates dependent on strain magnitude. Finally, macroscopic, shape-changing polymer-composite materials are developed to improve the current functionality of breast biopsy markers. This shape change provides a means to prevent marker migration from its intended site—a current clinical problem. In summary, mechanoresponsive systems are described, ranging from the nano- to macroscopic scale, for applications in drug delivery and biomedical devices. These studies add to the nascent field of mechanoresponsive biomedical materials and the arsenal of drug delivery techniques required to combat cancer and other medical ailments.2017-10-27T00:00:00

Adipogenesis within a Hollow Fiber-Based, Three-Dimensional Dynamic Perfusion Bioreactor

Adipose-derived stem cells (ASCs) represent a promising cell source in the field of tissue engineering and regenerative medicine. Due to the wide availability and multipotent ability of ASCs to differentiate into tissues such as bone, cartilage, muscle, and adipose, ASCs may serve a wide variety of regenerative medicine applications. Accordingly, ASCs have been utilized in studies addressing osteoarthritis, diabetes mellitus, heart disease, and soft tissue regeneration and reconstruction after mastectomy and facial trauma. Traditional, static, two-dimensional cell culture of ASCs do not allow for mature adipocyte differentiation or long-term maintenance of adipocytes in vitro. In order to study metabolic diseases, such as type II diabetes mellitus, a three-dimensional scaffold for in vitro adipocyte maintenance is necessary. In collaboration with the Bioreactor Laboratory at the McGowan Institute for Regenerative Medicine, our laboratory has developed the use of a hollow fiber-based bioreactor for three-dimensional, dynamic perfusion of ASCs and adipose tissue formation ex vivo, creating a stable system in which long-term culture of adipocytes is possible, providing a model useful for potential drug discovery and tissue engineering applications, specifically those addressing type II diabetes mellitus. The studies presented in this dissertation aim to assess metabolic activity and differentiation of ASCs from patients with or without type II diabetes in the bioreactor system; engineer a long-term culture environment relevant to physiological type II diabetic and non-diabetic conditions ex vivo; optimize tissue growth homogeneity; enhance adipogenesis within the bioreactor culture with the use of a decellularized adipose extracellular matrix (ECM) hydrogel. ASCs derived from patients with type II diabetes at time of isolation were found to behave metabolically similar and appear architecturally comparable to those derived from patients without type II diabetes mellitus when differentiated and maintained as adipocytes in the bioreactor system. When cultured at a physiologically relevant glucose level matching that of healthy patients or patients with type II diabetes, ASCs were able to proliferate, differentiate into adipocytes, and be maintained within the bioreactor system for at least one week. A decellularized adipose ECM hydrogel was established and applied to the bioreactor cultures; however, due to technical challenges, no firm conclusions can be made. The microenvironment by which ASCs are surrounded is critical for cell differentiation and growth. Engineering and control of such microenvironment is possible within the hollow fiber-based, three-dimensional, dynamic perfusion bioreactor culture system, proving to be a promising model for potential drug discovery and therapeutics. Future directions include further evaluation of ASC differentiation and adipocyte metabolism within type II diabetic environments, application of established decellularized adipose ECM hydrogels to wound healing treatments and adipose graft volume retention