Polymer- and Hybrid-Based Biomaterials for Interstitial, Connective, Vascular, Nerve, Visceral and Musculoskeletal Tissue Engineering

In this review, materials based on polymers and hybrids possessing both organic and inorganic contents for repairing or facilitating cell growth in tissue engineering are discussed. Pure polymer based biomaterials are predominantly used to target soft tissues. Stipulated by possibilities of tuning the composition and concentration of their inorganic content, hybrid materials allow to mimic properties of various types of harder tissues. That leads to the concept of “one-matches-all” referring to materials possessing the same polymeric base, but different inorganic content to enable tissue growth and repair, proliferation of cells, and the formation of the ECM (extra cellular matrix). Furthermore, adding drug delivery carriers to coatings and scaffolds designed with such materials brings additional functionality by encapsulating active molecules, antibacterial agents, and growth factors. We discuss here materials and methods of their assembly from a general perspective together with their applications in various tissue engineering sub-areas: interstitial, connective, vascular, nervous, visceral and musculoskeletal tissues. The overall aims of this review are two-fold: (a) to describe the needs and opportunities in the field of bio-medicine, which should be useful for material scientists, and (b) to present capabilities and resources available in the area of materials, which should be of interest for biologists and medical doctors.</jats:p

Biomedical applications of photochemistry

Photochemistry is the study of photochemical reactions between light and molecules. Recently, there have been increasing interests in using photochemical reactions in the fields of biomaterials and tissue engineering. This work revisits the components and mechanisms of photochemistry and reviews biomedical applications of photochemistry in various disciplines, including oncology, molecular biology, and biosurgery, with particular emphasis on tissue engineering. Finally, potential toxicities and research opportunities in this field are discussed. © 2010 Mary Ann Liebert, Inc.published_or_final_versio

Progress in Gelatin as Biomaterial for Tissue Engineering

Tissue engineering has become a medical alternative in this society with an ever-increasing lifespan. Advances in the areas of technology and biomaterials have facilitated the use of engineered constructs for medical issues. This review discusses on-going concerns and the latest developments in a widely employed biomaterial in the field of tissue engineering: gelatin. Emerging techniques including 3D bioprinting and gelatin functionalization have demonstrated better mimicking of native tissue by reinforcing gelatin-based systems, among others. This breakthrough facilitates, on the one hand, the manufacturing process when it comes to practicality and cost-effectiveness, which plays a key role in the transition towards clinical application. On the other hand, it can be concluded that gelatin could be considered as one of the promising biomaterials in future trends, in which the focus might be on the detection and diagnosis of diseases rather than treatment.This research was funded by the Spanish Ministry of Economy, Industry, and Competi- tiveness (PID2019-106094RB-I00/AEI/10.13039/501100011033) and the Basque Government who awarded Ph.D. grants (I.L. PRE_2021_2_0023; I.E. PRE_2021_2_0021)

Genetically engineered hydrogels based on elastin-like recombinamers for cardiovascular applications

Tissue engineering and regenerative medicine (TERM) is a prominent field of research that aims to repair or replace damaged tissues or organs, by the development of scaffolds with essential features, such as biocompatibility and functionality. Nowadays, recombinant polypeptides arise as promising candidates due to their tunability at the genetic level, affording exquisite control over the final physico-chemical properties and bioactivities. In particular, elastin-like recombinamers (ELRs) are genetically engineered polypeptides based on the repetition of the pentapeptide Val-Pro-Gly-X-Gly, found in the hydrophobic domains of tropoelastin, where X can be any amino acid except L-proline. These, ELRs exhibit a reversible phase transition in aqueous environments and their recombinant nature allows the inclusion of specific epitopes, such as cell adhesion, proteolytic sequences, and biologically active molecules such as growth factors. Interestingly, they can be chemically modified to obtain covalently cross-linked hydrogels through orthogonal and cytocompatible &#8220;click chemistry&#8221; reactions. The first chapter of this thesis is dedicated to the spatiotemporal control of angiogenesis, which has been proven essential for the correct integration and long-term stability of the implant. To this end, we designed a three-dimensional (3D) model consisting of a coaxial binary ELR tubular construct that displays proteolytic sequences with fast and slow cleavage kinetics towards the urokinase plasminogen activator protease on its inner and outer part respectively. The ELRs further included the universal cell-adhesion domain (RGD) and a VEGF-mimetic tethered peptide (QK) to induce angiogenesis. In vitro studies evidenced the effect of the QK peptide on endothelial cell extension and anastomosis. The subcutaneous implantation of the 3D models in mice showed a guided cell infiltration and capillary formation in the pre-designed spatiotemporal arrangement of the construct. Furthermore, the ELR hydrogels induced a mild macrophage response that resolved over time, supporting the potential integration of the resorbable scaffold within the host tissue. The second chapter study the preferential guidance of angiogenesis and neurogenesis in a spatiotemporal manner. In particular, we designed a 3D model ELR scaffold comprising two internal cylinders, with the pro-angiogenic peptide (QK) in one of them, and the neuronal cell adhesive peptide (IKVAV) in the vicinal one, both covalently tethered. In addition, these cylinders contain proteolytic sequences with fast cleavage kinetics towards the urokinase plasminogen activator enzyme and RGD cell adhesive domains. On the other hand, the outer part displays a slow-resorbable or non-protease-sensitive ELR hydrogel. In vitro studies demonstrated the effect of IKVAV epitope on neurite extension. The subcutaneous implantation of the 3D model ELR constructs in mice showed a guided cell infiltration accompanied by preferential angiogenesis or innervation on the respective QK and IKVAV containing cylinders, with a faster integration within the host tissue for the slow-resorbable scaffold. The third chapter describes the development of a ready-to-use bi-leaflet transcatheter venous valve for the treatment of chronic venous insufficiency (CVI), a leading worldwide vascular disease. For this purpose, we combined (i) ELRs, (ii) a textile mesh reinforcement and (iii) a bioabsorbable magnesium stent. Burst strength analysis demonstrated mechanical properties suitable for vascular pressures, whereas equibiaxial analysis confirmed the anisotropic performance equivalent to the native saphenous vein valves. In vitro studies identified the non-thrombogenic, minimal hemolysis and self-endothelialization properties endowed by the ELR hydrogel. The hydrodynamic testing under pulsatile conditions revealed minimal regurgitation (< 10%) and pressure drop (< 5 mmHg) in accordance with values stated for functional venous valves, and no stagnation points. Furthermore, in vitro simulated transcatheter delivery showed the ability to withstand the implantation procedure. In summary, the thesis presented herein provide new insights in the design and development of novel ELR-forming hydrogels to be used in tissue engineering and regenerative medicine applications.La ingeniería de tejidos y la medicina regenerativa (TERM) es un campo de investigación cuyo objetivo es reparar o reemplazar tejidos u órganos dañados, mediante el desarrollo de andamios biocompatibiles y funcionalizados. Hoy en día, los polipéptidos recombinantes, permiten un control exquisito sobre las propiedades fisicoquímicas y bioactividades. En particular, los elastin-like recombinamers (ELRs) son polipéptidos modificados genéticamente basados en la repetición del pentapéptido Val-Pro-Gly-X-Gly, que se encuentra en los dominios hidrófobos de la tropoelastina, donde X puede ser cualquier aminoácido excepto L-prolina. Estos ELR exhiben una transición de fase reversible en medios acuosos y su naturaleza recombinante permite la inclusión de epítopos específicos, como la adhesión celular, secuencias proteolíticas y moléculas bioactivas como factores de crecimiento. Curiosamente, pueden modificarse químicamente para obtener hidrogeles entrecruzados covalentemente a través de reacciones de "química de clic" ortogonales y citocompatibles. El primer capítulo está dedicado al control espaciotemporal de la angiogénesis, la cual es fundamental para la correcta integración y estabilidad del implante. Para ello, diseñamos un modelo tridimensional (3D) que consiste en una construcción binaria coaxial de hidrogeles de ELR, que lleva secuencias proteolíticas con cinética de escisión rápida y lenta sensibles a la proteasa del activador del plasminógeno tipo uroquinasa (uPA) en su parte interna y externa respectivamente, y un péptido mimético de VEGF (QK) anclado para inducir la angiogénesis. Los estudios in vitro evidenciaron el efecto del péptido QK sobre la extensión y anastomosis de las células endoteliales. La implantación subcutánea del modelo 3D en ratones mostró una infiltración celular guiada. Además, los hidrogeles ELR indujeron una respuesta leve de macrófagos que se resolvió con el tiempo, lo que respalda la integración de estos andamios reabsorbibles. El segundo capítulo estudia la orientación preferencial de la angiogénesis y la neurogénesis de manera espaciotemporal. En particular, diseñamos un modelo 3D de ELR que comprende dos cilindros internos, con el péptido proangiogénico (QK) en uno de ellos, y el péptido adhesivo de células neuronales (IKVAV) en el vecinal, ambos unidos covalentemente. Además, estos cilindros contienen secuencias proteolíticas con una cinética de escisión rápida frente a la enzima uPa y los dominios adhesivos RGD. Por otro lado, la parte exterior presenta un hidrogel ELR de reabsorción lenta o no sensible a las proteasas. Los estudios in vitro demostraron el efecto del epítopo IKVAV sobre la extensión de axones. La implantación subcutánea de las construcciones en ratones mostró una infiltración celular guiada acompañada de angiogénesis o inervación preferencial en los respectivos, con una integración más rápida dentro del tejido hospedador para el andamio con reabsorción lenta. El tercer capítulo describe el desarrollo de una válvula venosa transcatéter biválvula lista para usar para el tratamiento de la insuficiencia venosa crónica (IVC), una enfermedad vascular predominante en todo el mundo. Para ello, combinamos (i) ELR, (ii) un refuerzo de malla textil y (iii) un stent de magnesio bioabsorbible. El análisis de resistencia a rotura demostró propiedades mecánicas adecuadas para las presiones vasculares, mientras que el análisis equibiaxial confirmó el rendimiento anisotrópico equivalente a las válvulas de vena safena nativa. Los estudios in vitro identificaron las propiedades no trombogénicas, de hemólisis mínima y de autoendotelización que otorga el hidrogel ELR. Las pruebas hidrodinámicas en condiciones pulsátiles revelaron regurgitación mínima (< 10 %) y caída de presión (< 5 mmHg) de acuerdo con los valores establecidos para válvulas venosas funcionales y sin puntos de estancamiento. Además, el suministro transcatéter simulado in vitro mostró la capacidad de soportar el procedimiento de implantación. En resumen, la tesis presentada proporciona nuevos conocimientos en el diseño y desarrollo de nuevos hidrogeles ELR para su uso en ingeniería de tejidos y medicina regenerativa.Escuela de DoctoradoDoctorado en Físic

Engenharia de estruturas semelhantes a capilares incorporadas em hidrogéis para cultura de células 3D

Nowadays, the biggest challenge in tissue engineering consists in developing structures and in the application of strategies to emulate the anatomical and cellular complexity and vascularization of native tissues to maintain cell viability and functionality. The presence of functional blood vessel networks is essential to ensure adequate nutrient flow and oxygen diffusion throughout the support structure, two key requirements for maintaining cell viability. This work aimed to develop a complex in vitro model that mimics the native vascular network. To this end, a multilayered membrane made of six bilayers of chitosan (CHI)/alginate (ALG) or CHI/ALG-RGD (tripeptide of Arginine (R)-Glycine (G)- Aspartic acid (D) responsible for the cellular adhesion to the extracellular matrix (ECM)) were produced via Layer-by-Layer (LbL) assembly technology on the ALG printed structures. The ALG structures coated with the multilayered membranes were embedded in xanthan gum, chemically modified with methacrylated groups in order to obtain a mechanically robust hydrogel structure after photocrosslinking by UV light exposure. The liquification of the ALG printed structures, coated with the CHI/ALG, CHI/ALG-RGD or without the multilayers membranes, with ethylenediaminetetraacetic acid (EDTA), led to the formation of microchannels in which human umbilical vein endothelial cells (HUVECs) were cultured for 24 hours. The obtained results demonstrate that the microchannels encompassing CHI/ALG-RGD multilayered membranes contributed to a larger cellular adhesion, demonstrating their potential to be applied in tissue engineering and regenerative medicine strategies.Atualmente, o maior desafio em engenharia de tecidos consiste no desenvolvimento de estruturas e aplicação de estratégias que visem mimetizar a complexidade anatómica e celular, assim como a vascularização de tecidos nativos, de forma a manter a viabilidade e funcionalidade das células. A presença de estruturas funcionais à base de vasos sanguíneos é essencial para garantir o fluxo adequado de nutrientes, assim como a difusão de oxigénio em toda a estrutura de suporte, dois requisitos essenciais para manter a viabilidade celular. Este trabalho teve como objetivo desenvolver um modelo complexo in vitro que mimetize a rede vascular nativa. Com esse intuito, membranas multicamadas compreendendo seis bicamadas de quitosana (CHI)/alginato (ALG) e CHI/ALG-RGD (tripéptido de Arginina (R)-Glicina (G)-Ácido aspártico (D) responsável pela adesão de células à matriz extracelular) foram produzidas, via tecnologia de deposição camada-a-camada (do inglês Layer-by-Layer assembly technology), em estruturas impressas de ALG. As fibras de ALG revestidas com os filmes multicamadas foram embebidas em goma xantana, quimicamente modificada com grupos metacrilatos, de modo a obter uma estrutura de hidrogel mecanicamente robusta após foto-reticulação por ação da luz UV. A liquefação das estruturas impressas de ALG, contendo as multicamadas de CHI/ALG ou CHi/ALG-RGD, com ácido etilenodiamino tetra-acético (EDTA), levou à formação de microcanais nos quais se cultivaram células endoteliais humanas, extraídas da veia umbilical durante 24 horas. Os resultados obtidos demonstraram que os microcanais compreendendo as membranas multicamadas à base de CHI/ALG-RGD contribuíram para uma maior adesão celular, demonstrando o seu potencial para estratégias de engenharia de tecidos e medicina regenerativa.Mestrado em Biotecnologi

Catechol-functionalized hyaluronic acid and its application in tissue engineering

Biomaterials play pivotal roles in tissue engineering, as they can be used to produce scaffolds for tissue formation and serve as functional carriers for drug or cell delivery. The ability to support cell adhesion and tissue adhesion are critical properties of biomaterials to facilitate cell function and integration between implants and host tissues. Mussels can strongly adhere to various substrates due to the abundant catechol groups in the foot proteins. Catechol is reactive with both organic and inorganic substances, which has inspired research into developing catechol-functionalized materials for multiple applications. The adhesion to organic surfaces such as tissues makes catechol-functionalized materials attractive as tissue adhesive. Moreover, the versatile chemistry of catechol provides multiple strategies for producing hydrogels for tissue engineering. The aim of this project is to utilize the catechol chemistry to develop catechol-functionalized biomaterials and exploit their applications in tissue engineering. Hyaluronic acid (HA) was chosen as the backbone for catechol conjugation mainly attributed to its multiple biological activities. The crosslinking conditions of catechol-functionalized HA (HACA) were optimized, the adhesion, swelling and degradation behaviour of the crosslinked hydrogels were characterized. The HACA-based material demonstrated good adhesion to hydrogels derived from collagen and gelatin that act as a simplified soft tissue model, and to porcine skin tissue. Moreover, it supported culture of a human umbilical vein endothelial cell line (HUV-EC-C) with high cell viability and formation of capillary-like structure. This may bring added benefit by means of promoting angiogenesis, therefore promoting the integration between host tissue and implant. The results indicate that the HACA-based material could be a promising tissue adhesive for multiple internal uses

Coalescence of ECM and chitosan biomaterials for an advanced sutureless technology

The ideal wound closure device should restore the original integrity of the tissue, offer easy application and seamless, fluid-tight seal. Commonly used wound closure devices including sutures, staples and tissue adhesives do not offer effective sealing of the wound and possess a range of associated disadvantages including dehiscence, infection, toxicity and iatrogenic related trauma. Laser tissue welding has been proposed as an alternative and is becoming increasingly popular. This technique uses laser irradiation to initiate chemical reactions in a target material, thereby activating chemical components within the material to form an immediate seal. However, laser tissue welding is reliant upon temperatures exceeding the collagen denaturation point and is associated with some tissue damage. SurgiLux is a chitosan-based thin film surgical adhesive that relies upon laser irradiation to increase the strength of chitosan binding to tissue without thermal damage to tissue and avoiding many of the disadvantages of current wound closure devices. Previous in vitro and in vivo studies of SurgiLux has demonstrated the potential of SurgiLux in sutureless repair of peripheral nerves. Recent approaches to regenerative medicine and tissue engineering involve the use of decellularised extracellular matrix as biological scaffolds to augment the formation of new functional tissue and facilitate successful tissue reconstruction. The aim of the work reported here was to combine SurgiLux with an extracellular matrix scaffold derived from porcine urinary bladder matrix to potentially improve the capacity of the SurgiLux technology to enhance wound healing by promoting functional tissue regeneration, for potential applications in peripheral nerve repair. The bio-scaffold was incorporated into the SurgiLux film in a variety of ways; bio-scaffold embedded into SurgiLux had a greater tensile strength (32.4 ± 5.2 MPa), crystallinity (12.1 ± 1.3 %) and hydrophilicity (75.0 ± 2.0)° than the chitosan adhesive alone (8.5 ± 3.1 MPa, 10.7 ± 1.2% crystallinity, Ө = 98.1 ± 2.03°). Tissue adhesion strengths using these hybrid biomaterials were maintained at ~15 kPa compared to 3 kPa for fibrin glue. Furthermore, histological analysis demonstrated that laser irradiation of the UBM-SurgiLux adhesive caused no thermal damage to tissue. In vitro biocompatibility of the composite films was assessed by examining their influence on the proliferation and health of olfactory ensheathing cells and human monocyte-derived macrophages. Incorporation of the bio-scaffold into the SurgiLux films increased the attachment and proliferation of olfactory ensheathing cells and decreased the cytotoxicity of the films. Similarly, while chitosan films induced a cell population to undergo early apoptotic activation, the composite films apparently increased biocompatibility, preventing the cells from undergoing necrosis. Similarly, while SurgiLux showed a significantly reduced macrophage response compared to chitosan film, introduction of the bio-scaffold into the SurgiLux reduced their response further. A quantitative real time PCR approach was undertaken to identify polarised macrophage phenotype, M1 (pro-inflammatory) and M2 (anti-inflammatory) through the detection of specific cytokines expressed by the macrophages. Reduced cell spread (6.2e3 ± 7.0e2 μm2) and lack of foreign body giant cell formation lead to significantly reduced expression of M1 markers, IL-23p19, IL-12p40 and IL-12p35; thereby suggesting the presence of an alternative anti-inflammatory, tissue remodelling pathway. A protein expression profile of the UBM scaffold was generated to identify novel proteins within the UBM via an advanced mass spectrometry methodology. A total of 129 proteins were identified with the majority of these revealing a role in maintaining cell structure (19%) and adhesion (13%), while the smallest groups (1 %) had remodelling and stimulatory roles. A number of growth promoting proteins including galectins 1 and 7, obscurin, fibulin and have been identified that may have enhanced cellular proliferation on the UBM-SurgiLux composite scaffolds compared to chitosan films alone. UBM also contains proteins that have neurotrophic, anti-angiogenic, tumour suppressor activity and proteins known to promote tissue remodelling and morphogenesis. Therefore, coalescence of the bio-scaffold within SurgiLux matrix resulted in a surgical adhesive with enhanced biocompatibility and reduced cytotoxicity compared to chitosan films. The results suggest that the unique combination of extracellular matrix bio-scaffold with SurgiLux technology has the potential to promote functional tissue regeneration leading to enhanced sutureless nerve repair