A multilayered microfluidic blood vessel-like structure

There is an immense need for tissue engineered blood vessels. However, current tissue engineering approaches still lack the ability to build native blood vessel-like perfusable structures with multi-layered vascular walls. This paper demonstrated a new method to fabricate tri-layer biomimetic blood vessel-like structures on a microfluidic platform using photocrosslinkable gelatin hydrogel. The presented method enables fabrication of physiological blood vessel-like structures with mono-, bi- or tri-layer vascular walls. The diameter of the vessels, the total thickness of the vessel wall and the thickness of each individual layer of the wall were independently controlled. The developed fabrication process is a simple and rapid method, allowing the physical fabrication of the vascular structure in minutes, and the formation of a vascular endothelial cell layer inside the vessels in 3–5 days. The fabricated vascular constructs can potentially be used in numerous applications including drug screening, development of in vitro models for cardiovascular diseases and/or cancer metastasis, and study of vascular biology and mechanobiology.American University of Beirut (startup grant and University Research Board grant)National Council for Scientific Research (Lebanon)National Science Foundation (U.S.) (EFRI-1240443)Immodgel (602694)National Institutes of Health (U.S.) (EB012597, AR057837, DE021468, HL099073, AI105024, AR063745)National Institute of General Medical Sciences (U.S.) ( Award Number P20GM103638-04)King Abdulaziz City for Science and Technology (Grant No. 12-MED3096-3

Multiscale multifactorial approaches for engineering tendon substitutes

The physiology of tendons and the continuous strains experienced daily make tendons very prone to injury. Excessive and prolonged loading forces and aging also contribute to the onset and progression of tendon injuries, and conventional treatments have limited efficacy in restoring tendon biomechanics. Tissue engineering and regenerative medicine (TERM) approaches hold the promise to provide therapeutic solutions for injured or damaged tendons despite the challenging cues of tendon niche and the lack of tendon-specific factors to guide cellular responses and tackle regeneration. The roots of engineering tendon substitutes lay in multifactorial approaches from adequate stem cells sources and environmental stimuli to the construction of multiscale 3D scaffolding systems. To achieve such advanced tendon substitutes, incremental strategies have been pursued to more closely recreate the native tendon requirements providing structural as well as physical and chemical cues combined with biochemical and mechanical stimuli to instruct cell behavior in 3D architectures, pursuing mechanically competent constructs with adequate maturation before implantation.Authors acknowledge the project “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marinederived biomaterials and stem cells,” supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Authors acknowledge the H2020 Achilles Twinning Project No. 810850, and also the European Research Council CoG MagTendon No. 772817, and the FCT Project MagTT PTDC/CTM-CTM/ 29930/2017 (POCI-01-0145-FEDER-29930

Biomaterials as Tendon and Ligament Substitutes: Current Developments

Tendon and ligament have specialized dynamic microenvironment characterized by a complex hierarchical extracellular matrix essential for tissue functionality, and responsible to be an instructive niche for resident cells. Among musculoskeletal diseases, tendon/ligament injuries often result in pain, substantial tissue morbidity, and disability, affecting athletes, active working people and elder population. This represents not only a major healthcare problem but it implies considerable social and economic hurdles. Current treatments are based on the replacement and/or augmentation of the damaged tissue with severe associated limitations. Thus, it is evident the clinical challenge and emergent need to recreate native tissue features and regenerate damaged tissues. In this context, the design and development of anisotropic bioengineered systems with potential to recapitulate the hierarchical architecture and organization of tendons and ligaments from nano to macro scale will be discussed in this chapter. Special attention will be given to the state-of-the-art fabrication techniques, namely spinning and electrochemical alignment techniques to address the demanding requirements for tendon substitutes, particularly concerning the importance of biomechanical and structural cues of these tissues. Moreover, the poor innate regeneration ability related to the low cellularity and vascularization of tendons and ligaments also anticipates the importance of cell based strategies, particularly on the stem cells role for the success of tissue engineered therapies. In summary, this chapter provides a general overview on tendon and ligaments physiology and current conventional treatments for injuries caused by trauma and/or disease. Moreover, this chapter presents tissue engineering approaches as an alternative to overcome the limitations of current therapies, focusing on the discussion about scaffolds design for tissue substitutes to meet the regenerative medicine challenges towards the functional restoration of damaged or degenerated tendon and ligament tissues.Portuguese Foundation for Science and Technology for the post-doctoral grant (SFRH/BPD/111729/2015) and for the projects Recognize (UTAP-ICDT/CTM-BIO/0023/2014) and POC I-01-0145-FEDER-007

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell’s perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy

Ultrasound Imaging Techniques for Spatiotemporal Characterization of Composition, Microstructure, and Mechanical Properties in Tissue Engineering

Ultrasound techniques are increasingly being used to quantitatively characterize both native and engineered tissues. This review provides an overview and selected examples of the main techniques used in these applications. Grayscale imaging has been used to characterize extracellular matrix deposition, and quantitative ultrasound imaging based on the integrated backscatter coefficient has been applied to estimating cell concentrations and matrix morphology in tissue engineering. Spectral analysis has been employed to characterize the concentration and spatial distribution of mineral particles in a construct, as well as to monitor mineral deposition by cells over time. Ultrasound techniques have also been used to measure the mechanical properties of native and engineered tissues. Conventional ultrasound elasticity imaging and acoustic radiation force imaging have been applied to detect regions of altered stiffness within tissues. Sonorheometry and monitoring of steady-state excitation and recovery have been used to characterize viscoelastic properties of tissue using a single transducer to both deform and image the sample. Dual-mode ultrasound elastography uses separate ultrasound transducers to produce a more potent deformation force to microscale characterization of viscoelasticity of hydrogel constructs. These ultrasound-based techniques have high potential to impact the field of tissue engineering as they are further developed and their range of applications expands.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140238/1/ten.teb.2015.0453.pd

Osteochondral tissue repair in osteoarthritic joints: clinical challenges and opportunities in tissue engineering

Osteoarthritis (OA), identified as one of the priorities for the Bone and Joint Decade, is one of the most prevalent joint diseases, which causes pain and disability of joints in the adult population. Secondary OA usually stems from repetitive overloading to the osteochondral (OC) unit, which could result in cartilage damage and changes in the subchondral bone, leading to mechanical instability of the joint and loss of joint function. Tissue engineering approaches have emerged for the repair of cartilage defects and damages to the subchondral bone in the early stages of OA and have shown potential in restoring the joint’s function. In this approach, the use of three-dimensional scaffolds (with or without cells) provides support for tissue growth. Commercially available OC scaffolds have been studied in OA patients for repair and regeneration of OC defects. However, none of these scaffolds has shown satisfactory clinical results. This article reviews the OC tissue structure and the design, manufacturing and performance of current OC scaffolds in treatment of OA. The findings demonstrate the importance of biological and biomechanical fixations of OC scaffolds to the host tissue in achieving an improved cartilage fill and a hyaline-like tissue formation. Achieving a strong and stable subchondral bone support that helps the regeneration of overlying cartilage seems to be still a grand challenge for the early treatment of OA

Cápsulas liquefeitas contendo micropartículas com rigidez variável para controlar diferenciação celular

Tissue engineering and regenerative medicine (TERM) strategies make use of selective cell differentiation to engineer biomimetic tissues through a series of factors, with a recent focus on mechanotransduction-based differentiation. Such mechanobiology-based TERM (or Mech-TERM) strategies make use of the mechanical forces in any given system to steer tissue formation towards desired pathways. This is performed by attempting to emulate the forces present in the tissue that we want to replicate, bringing as such an entirely new layer of biomimicry to TE systems. One such form of systems is bioencapsulation with liquefied capsules, due to its highly customizable nature and potential for application through minimally invasive procedures. This type of system also ensures high cell viability and proliferation, as well as immunoprotection. In this work, human adipose stem cells (hASCs) were co-encapsulated with microparticles of distinct stiffnesses, acting as 3D scaffolds for cellular adhesion and development. Our hypothesis is that scaffolds with stiffnesses emulating those of bone or articular cartilage will be able to guide cell differentiation into osteogenic or chondrogenic lineages, respectively, through the process of mechanotransduction. For that, hASCs were co-encapsulated with gelatin-grafted polycaprolactone (PCL) microparticles (STIFF+ capsules) or with gelatin microparticles (STIFF- capsules) and cultured for 28 days in basal (BAS), osteogenic (OST), and chondrogenic (CHO) differentiation media, followed by a series of analysis to determine the extent of cell viability, proliferation, and differentiation into the osteogenic and chondrogenic lineages. This system showed that high cell viability and proliferation were overall maintained, and that traces of osteogenic and chondrogenic differentiation were observed in both capsule types. Hydroxyapatite (HA) formation was detected in STIFF+ capsules cultured in BAS and OST media, and in STIFF- capsules in OST medium, at 21 days. Collagen type II was detected in both capsule types and in all media, in different amounts, at day 28. Alkaline phosphatase (ALP) activity and sulfated glycosaminoglycans (sGAG) content profiles overall indicated that STIFF+ and STIFF- capsules had, respectively, favored osteogenic and chondrogenic differentiation. Future studies involving more specific assays, such as identifying and/or quantifying expression of key osteogenic and chondrogenic genes and pathways in hASCs, are suggested to fully validate this hypothesis.Estratégias de engenharia de tecidos e medicina regenerativa fazem uso de diferenciação celular seletiva para engenhar tecidos biomiméticos através de vários factores, com um foco recente na diferenciação por mecanotransdução. Tais estratégias com base em mecanobiologia fazem uso de forças mecânicas em qualquer dado sistema para guiar a formação de tecidos por vias desejadas. Isto é alcançado tentando simular as forças presentes no tecido que queremos replicar, trazendo dessa forma uma nova camada de biomimetismo a sistemas de engenharia de tecidos. Um destes sistemas é o bioencapsulamento com cápsulas liquefeitas, devido à sua natureza altamente customizável e ao seu potencial para aplicação através de procedimentos minimamente invasivos. Este género de sistema também assegura viabilidade e proliferação celular elevadas, juntamente com proteção contra respostas imunitárias do paciente. Neste trabalho, células estaminais mesenquimais isoladas do tecido adiposo (hASCs) foram co-encapsuladas com micropartículas, de rigidez distinta. As micropartículas têm o objetivo de fornecer pontos para adesão e desenvolvimento celular. Numa tentativa de determinar os efeitos da rigidez na diferenciação celular através de mecanotransdução, foram desenvolvidas micropartículas de rigidez distinta. Para tal, hASCs foram co-encapsuladas com micropartículas de policaprolactona (PCL) com um revestimento de gelatina (cápsulas STIFF+) ou com micropartículas de gelatina (cápsulas STIFF-). As diferentes cápsulas foram incubadas durante 28 dias em meio de cultura basal (BAS), ou com fatores de diferenciação osteogénica (OST) ou condrogénica (CHO). Diferentes análises foram efetuadas para determinar a extensão de viabilidade e proliferação celular, bem como de diferenciação em linhagens osteogénicas e condrogénicas. Este sistema demonstrou manter elevada viabilidade e proliferação celulares em geral, e diferenciações osteogénicas e condrogénicas foram observadas em ambos os tipos de cápsulas. Formação de hydroxyapatite (HA) foi detetada em cápsulas STIFF+ em meio BAS e OST, e em cápsulas STIFF- em meio OST, aos 21 dias. Colagénio tipo II foi detetado em ambos os tipos de cápsulas e em todos os meios, em quantidades diferentes, aos 28 dias. Perfis de actividade de fosfatase alcalina (ALP) e conteúdos de glicoasminoglicanos sulfatados (sGAG) indicaram em geral que as cápsulas STIFF+ e STIFF-, respectivamente, favoreceram mais a diferenciação osteogénica e condrogénica. Estudos futuros envolvendo análises mais específicas, tais como identificar e/ou quantificar a expressão de genes e vias osteogénicas e condrogénicas chave em hASCs, são sugeridos para validar completamente esta hipótese.Mestrado em Bioquímic

Enhancing Biological and Biomechanical Fixation of Osteochondral Scaffold: A Grand Challenge

Osteoarthritis (OA) is a degenerative joint disease, typified by degradation of cartilage and changes in the subchondral bone, resulting in pain, stiffness and reduced mobility. Current surgical treatments often fail to regenerate hyaline cartilage and result in the formation of fibrocartilage. Tissue engineering approaches have emerged for the repair of cartilage defects and damages to the subchondral bones in the early stage of OA and have shown potential in restoring the joint's function. In this approach, the use of three-dimensional scaffolds (with or without cells) provides support for tissue growth. Commercially available osteochondral (OC) scaffolds have been studied in OA patients for repair and regeneration of OC defects. However, some controversial results are often reported from both clinical trials and animal studies. The objective of this chapter is to report the scaffolds clinical requirements and performance of the currently available OC scaffolds that have been investigated both in animal studies and in clinical trials. The findings have demonstrated the importance of biological and biomechanical fixation of the OC scaffolds in achieving good cartilage fill and improved hyaline cartilage formation. It is concluded that improving cartilage fill, enhancing its integration with host tissues and achieving a strong and stable subchondral bone support for overlying cartilage are still grand challenges for the early treatment of OA

Compact and tunable stretch bioreactor advancing tissue engineering implementation. Application to engineered cardiac constructs

Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro . In tissue engineering applications, bioreactors have become fundamental and effective tools for provid- ing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on de- cellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation

Development and Applications of Advanced Ultrasound Techniques for Characterization and Stimulation of Engineered Tissues

Mechanobiology is central in the development, pathology, and regeneration of musculoskeletal tissues, in which mechanical factors play important roles. Therefore, there is a need for methods to characterize the composition and mechanical properties of developing musculoskeletal tissues over time. Ultrasound elastographic techniques have been developed for noninvasive imaging of spatial heterogeneity in tissue stiffness. However, their application for quantitative assessment of tissue mechanical properties, especially viscoelastic properties, has not been exploited. Additionally, ultrasound energy may be used to apply mechanical stimulation to engineered constructs at the microscale, and thereby to enhance tissue regeneration. We have developed a multimode ultrasound viscoelastography (MUVE) system for assessing microscale mechanical properties of engineered hydrogels. MUVE uses focused ultrasound pulses to apply acoustic radiation force (ARF) to deform samples, while concurrently measuring sample dimensions using coaxial high frequency ultrasound imaging. We used MUVE to perform creep tests on agarose, collagen, and fibrin hydrogels of defined concentrations, as well as to monitor the mechanical properties of cell-seeded constructs over time. Local and bulk viscoelastic properties were extracted from strain-time curves through fitting of relevant constitutive models, showing clear differences between concentrations and materials. In particular, we showed that MUVE is capable of mapping heterogeneity of samples in 3D. Using inclusion of dense agarose microbeads within agarose, collagen and fibrin hydrogels, we determined the spatial resolution of MUVE to be approximately 200 μm in both the lateral and axial directions. Comparison of MUVE to nanoindentation and shear rheometry showed that our ultrasound-based technique was superior in generating consistent, microscale data, particularly for very soft materials. We have also adapted MUVE to generate localized cyclic compression, as a means to mechanically stimulate engineered tissue constructs at the microscale. Selected treatment protocols were shown to enhance the osteogenic differentiation of human mesenchymal stem cells in collagen-fibrin hydrogels. Constructs treated at 1 Hz at an acoustic pressure of 0.7 MPa for 30 minutes per day showed accelerated osteogenesis and increased mineralization by 10 to 30 percent, relative to unstimulated controls. In separate experiments, the ultrasound pulse intensity was increased over time to compensate for changes in matrix properties over time, and a 35 percent increase in mineralization was achieved. We also extended the application of a previously-developed spectral ultrasound imaging (SUSI) technique to an animal model for early detection of heterotopic ossification (HO). The quantitative information on acoustic scatterer size and concentration derived from SUSI was used to differentiate tissue composition in a burn/tenotomy mice model from the control model. Importantly, HO foci were detected as early as one week after injury using SUSI, which is 3-5 weeks earlier than when using conventional micro-computed tomography. Taken together, these results demonstrate that ultrasound-based techniques can non-invasively and quantitatively characterize viscoelastic properties of soft materials in 3D, as well as their composition over time. Ultrasound pulses can also be used to stimulate engineered constructs to promote musculoskeletal tissue formation. MUVE, SUSI, and ultrasound stimulation can be combined into an integrated system to investigate the roles of matrix composition, static mechanical environment, and dynamic mechanical stimuli in tissue regeneration, as well as the interactions of these factors and their evolution over time. Ultrasound-based techniques therefore have promising potential in noninvasively characterizing the composition and biomechanics, as well as providing mechanical intervention in native and engineered tissues as they develop over time.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144116/1/xho_1.pd