Hybrid bioprinting of chondrogenically induced human mesenchymal stem cell spheroids

To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 +/- 2.80 mu m, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)- and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids, gelMA is a promising material as it exhibits favorable properties in terms of printability and it supports the viability and chondrogenic phenotype of hBM-MSC microtissues. Moreover, it was shown that a lower hydrogel stiffness enhances further chondrogenic maturation after bioprinting

Bioink properties before, during and after 3D bioprinting

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction

Trends in the design and use of elastin-like recombinamers as biomaterials

Producción CientíficaElastin-like recombinamers (ELRs), which derive from one of the repetitive domains found in natural elastin, have been intensively studied in the last few years from several points of view. In this mini review, we discuss all the recent works related to the investigation of ELRs, starting with those that define these polypeptides as model intrinsically disordered proteins or regions (IDPs or IDRs) and its relevance for some biomedical applications. Furthermore, we summarize the current knowledge on the development of drug, vaccine and gene delivery systems based on ELRs, while also emphasizing the use of ELR-based hydrogels in tissue engineering and regenerative medicine (TERM). Finally, we show different studies that explore applications in other fields, and several examples that describe biomaterial blends in which ELRs have a key role. This review aims to give an overview of the recent advances regarding ELRs and to encourage further investigation of their properties and applications.Comisión Europea (project NMP-2014-646075)Ministerio de Economía, Industria y Competitividad (projects PCIN-2015-010 / MAT2016-78903-R / BES-2014-069763)Junta de Castilla y León (project VA317P18

Translational cell based therapies to repair the heart

Cardiovascular disease comprising of Coronary Artery Disease (CAD) and Valvular Heart Disease (VHD) represents the leading disease in western societies accounting for the death of numerous patients. CAD may lead to heart failure (HF) and despite the therapeutic options for HF which evolved over the past years, the incidence of HF is continuously increasing with a higher percentage of aged people. Similarly, an increase of VHD can be observed and although valve replacement represents the most common therapy strategy for VHD, approximately 30% of the treated patients are affected from prosthesis-related problems within 10 years. While mechanical valves require lifelong anticoagulation treatment, bioprosthetic valves present with continuous degeneration without the ability to grow, repair or remodel. The concept of regenerative medicine comprising of cell-based therapies, bio engineering technologies and hybrid solutions has been proposed as a promising next generation approach to address CAD and VHD. While myocardial cell therapy has been suggested to have a beneficial effect on the failing myocardium, heart valve tissue engineering has been demonstrated to be a promising concept to generate living, autologous heart valves with the capability to grow and to remodel which may be particularly beneficial for children. Although these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or has been too rapid and premature leaving many key questions unanswered. The aim of this thesis was the systematic development of translational, cell-based bio engineering concepts addressing CAD (part A) and VHD (part B) with a particular focus on minimally invasive, transcatheter-based implantation techniques. In the setting of myocardial regeneration, in the second chapter the intrinsic regenerative potential of the heart is investigated. Myocardial samples were harvested from all four chambers of the human heart and were assessed for resident stem/progenitor cell populations. The results demonstrated that BRCP+ cells can be detected within the human heart and that they were more abundant than their c-kit+ counterparts. In the non-ischemic heart they were preferentially located in the atria while following ischemia, their numbers were increased significantly in the left ventricle. There were no c-kit+/BCRP+ co-expressing stem/progenitor cell populations suggesting that these two markers are expressed by two distinct cell populations in the human heart. Although these results provided a valuable snapshot at cardiac progenitor cells after acute ischemia, the data also indicated that the absolute numbers of cells acquiring a myocardial phenotype are rather low and further effort is needed to upscale such cells into clinically relevant numbers. In chapter three, it is demonstrated that human bone marrow and adipose tissue derived mesenchymal stem cells can be efficiently isolated via minimally invasive procedures and expanded to clinically relevant numbers for myocardial cell therapy. Thereafter, these cells were tested in a uniquely developed intrauterine, fetal, preimmune ovine myocardial infarction model for the evaluation of human cell fate in vivo. After the successful intrauterine induction of acute myocardial infarction, the cells were intramyocardially transplanted and tracked using a multimodal imaging approach comprising MRI, Micro CT as well as in vitro analysis tools. The principal feasibility of intra-myocardial stem-cell transplantation following intra-uterine induction of myocardial infarction in the preimmune fetal sheep could be demonstrated suggesting this as a unique platform to evaluate human cell-fate in a relevant large animal-model without the necessity of immunosuppressive therapy. In chapter four, adipose tissue derived mesenchymal stem cells (ATMSCs) were processed to generate three dimensional microtissues (3D-MTs) prior to transplantation to address the important issue of cell retention and survival. Thereafter, the ATMSCs based 3D-MTs were transplanted into the healthy and infarcted porcine myocardium using a catheter-based, 3D electromechanical mapping guided approach. The previously used MRI based tracking concept was successfully translated into this preclinical model allowing for the in vivo monitoring of 3D-MTs. To address Valvular Heart Disease (part B), in chapter five, marrow stromal derived cells were used to develop a unique autologous, cell-based engineered heart valve in situ tissue engineering concept comprising of minimally-invasive techniques for both, cell harvest and valve implantation. Autologous marrow stromal derived cells were harvested, seeded onto biodegradable scaffolds and integrated into self-expanding nitinol stents, before they were transapically delivered into the pulmonary position of non-human primates within the same intervention while avoiding any in vitro bio-reactor period. The results of these experiments demonstrated the principal feasibility of generating marrow stromal cell-based, autologous, living tissue engineered heart valves (TEHV) and the transapical implantation in a one-step intervention. In chapter six, this concept was then successfully applied to the high-pressure system of the systemic circulation. After detailed adaption of the TEHV and stent design to the anatomic conditions of an orthotopic aortic valve, marrow stromal cell-based TEHV were implanted into the orthotopic aortic position. The implantation was successful and valve functionality was confirmed using fluoroscopy and trans-esophageal echocardiography. While displaying an ideal opening and closing behaviour with a sufficient co-aptation and a low pressure gradient, there were no signs of coronary occlusion or mal-perfusion. In conclusion, the results of this thesis represent a promising portfolio of translational concepts for cardiovascular regenerative medicine addressing CAD and VHD. In particular, it was demonstrated that mesenchymal stem cells / multipotent stromal derived cells represent a clinically relevant cell source for both myocardial regeneration and heart valve tissue engineering. It was shown that the preimmune fetal sheep myocardial infarction model represents a unique platform for the in vivo evaluation of human stem cells without the necessity of immunosuppressive therapy. Moreover, the concept of transcatheter based intramyocardial transplantation of mesenchymal stem cell-based 3D-MTs was introduced to enhance cellular retention and survival. Finally, in the setting of VHD it could be shown that marrow stromal cell based issue engineered heart valves can successfully generated and transapically implanted into the pulmonary and aortic position within a one-step intervention

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

3D-Bioprinting: A stepping stone towards enhanced medical approaches

In the past few decades, tissue engineering has been seen unprecedented escalation driving the field of artificial tissue and organ construct and brought metamorphosis in regenerative medicine. Prime advancement has been attained through the expansion of novel biomanufacturing approaches to devise and convene cells in three dimensions to fabricate tissue contrive. Accompaniment manufacturing differently known as 3D bioprinting is leading prime innovation in a number of applications in life sciences such as tissue and organ construct, personalized drug dosing, cancer model and heart tissue engineering. Overall, this review summarizes most prevalent bioprinting technologies; including laser-based bioprinting, extrusion bioprinting, injection bioprinting, stereolithography as well as biomaterial such as bioink. It also explores 3D industries, approaches such as Biomimicry, autonomous self-assembly, mini tissues and biomedical applications. Existing challenges that impede clinical mileage of bioprinting are also discussed along with future prospective.Keywords: Bioprinting, tissue engineering, tissue and organ construct, medicinal approac

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

Design and functional testing of a multichamber perfusion platform for three-dimensional scaffolds

Perfusion culture systems are widely used in tissue engineering applications for enhancing cell culture viability in the core of three-dimensional scaffolds. In this work, we present a multichamber confined-flow perfusion system, designed to provide a straightforward platform for three-dimensional dynamic cell cultures. The device comprises 6 culture chambers allowing independent and simultaneous experiments in controlled conditions. Each chamber consists of three parts: a housing, a deformable scaffold-holder cartridge, and a 7 mL reservoir, which couples water-tightly with the housing compressing the cartridge. Short-term dynamic cell seeding experiments were carried out with MC3T3-E1 cells seeded into polycaprolactone porous scaffolds. Preliminary results revealed that the application of flow perfusion through the scaffold favored the penetration of the cells to its interior, producing a more homogeneous distribution of cells with respect to dropwise or injection seeding methods. The culture chamber layout was conceived with the aim of simplifying the user operations under laminar flow hood and minimizing the risks for contamination during handling and operation. Furthermore, a compact size, a small number of components, and the use of bayonet couplings ensured a simple, fast, and sterility-promoting assembling. Finally, preliminary in vitro tests proved the efficacy of the system in enhancing cell seeding efficiency, opening the way for further studies addressing long-term scaffold colonization

Vascularization of tissue engineered cartilage - Sequential in vivo MRI display functional blood circulation

Establishing functional circulation in bioengineered tissue after implantation is vital for the delivery of oxygen and nutrients to the cells. Native cartilage is avascular and thrives on diffusion, which in turn depends on proximity to circulation. Here, we investigate whether a gridded three-dimensional (3D) bioprinted construct would allow ingrowth of blood vessels and thus prove a functional concept for vascularization of bioengineered tissue. Twenty 10 7 10 7 3-mm 3Dbioprinted nanocellulose constructs containing human nasal chondrocytes or cell-free controls were subcutaneously implanted in 20 nude mice. Over the next 3 months, the mice were sequentially imaged with a 7 T small-animal MRI system, and the diffusion and perfusion parameters were analyzed. The chondrocytes survived and proliferated, and the shape of the constructs was well preserved. The diffusion coefficient was high and well preserved over time. The perfusion and diffusion patterns shown by MRI suggested that blood vessels develop over time in the 3D bioprinted constructs; the vessels were confirmed by histology and immunohistochemistry. We conclude that 3D bioprinted tissue with a gridded structure allows ingrowth of blood vessels and has the potential to be vascularized from the host. This is an essential step to take bioengineered tissue from the bench to clinical practice

Nanowired Human Cardiac Spheroids for Cardiac Regenerative Medicine

3D scaffold-free spherical micro-tissue (spheroids) holds great potential in tissue engineering as building blocks to fabricate the functional tissues or organs in vitro. To date, agarose based hydrogel molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we developed an alginate-based, direct 3D mold-printing technology: 3D printing micro-droplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit micro-droplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D agarose hydrogel molds. Tissue spheroids composed of 50% human endothelial cells and 50% human smooth muscle cells were robotically dispensed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell-cell adhesion, tissue formation and maturation. The current inability to derive mature cardiomyocytes (CMs) from human pluripotent stem cells (hiPSC) has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of CMs derived from hiPSC. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids prepared by seeding CMs into agarose microwells were shown to improve cardiac functions. However, CMs within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here we show, for the first time, that incorporation of a trace amount (i.e., ~0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., \u3e55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (~0.5k, ~1k, ~3k, ~7k cells/spheroid). Spheroids with ~3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. We reasoned that the presence of e-SiNWs in the injectable spheroids improves their ability to receive exogenous electromechanical pacing from the host myocardium to enhance their integration with host tissue post-transplantation. In this study, we examined the cardiac biocompatibility of the e-SiNWs and cell retention, engraftment and integration after injection of the nanowired hiPSC cardiac spheroids into adult rat hearts. Our results showed that the e-SiNWs caused minimal toxicity to rat adult hearts after intramyocardial injection. Further, the nanowired spheroids were shown to significantly improve cell retention and engraftment, when compared to dissociated hiPSC-CMs and unwired spheroids. The 7-days-old nanowired spheroid grafts showed alignment with the host myocardium and development of sarcomere structures. The 28-days-old nanowired spheroid grafts showed gap junctions, mechanical junctions and vascular integration with host myocardium. Together, our results clearly demonstrate the remarkable potential of the nanowired spheroids as cell delivery vehicles to treat cardiovascular diseases