Image-based analysis of uniaxial ring test for mechanical characterization of soft materials and biological tissues.

Uniaxial ring test is a widely used mechanical characterization method for a variety of materials, from industrial elastomers to biological materials. Here we show that the combination of local material compression, bending, and stretching during uniaxial ring test results in a geometry-dependent deformation profile that can introduce systematic errors in the extraction of mechanical parameters. We identify the stress and strain regimes under which stretching dominates and develop a simple image-based analysis approach that eliminates these systematic errors. We rigorously test this approach computationally and experimentally, and demonstrate that we can accurately estimate the sample mechanical properties for a wide range of ring geometries. As a proof of concept for its application, we use the approach to analyze explanted rat vascular tissues and find a clear temporal change in the mechanical properties of these explants after graft implantation. The image-based approach can therefore offer a straightforward, versatile, and accurate method for mechanically characterizing new classes of soft and biological materials.

Computationally guided in-vitro vascular growth model reveals causal link between flow oscillations and disorganized neotissue.

Disturbed shear stress is thought to be the driving factor of neointimal hyperplasia in blood vessels and grafts, for example in hemodialysis conduits. Despite the common occurrence of neointimal hyperplasia, however, the mechanistic role of shear stress is unclear. This is especially problematic in the context of in situ scaffold-guided vascular regeneration, a process strongly driven by the scaffold mechanical environment. To address this issue, we herein introduce an integrated numerical-experimental approach to reconstruct the graft-host response and interrogate the mechanoregulation in dialysis grafts. Starting from patient data, we numerically analyze the biomechanics at the vein-graft anastomosis of a hemodialysis conduit. Using this biomechanical data, we show in an in vitro vascular growth model that oscillatory shear stress, in the presence of cyclic strain, favors neotissue development by reducing the secretion of remodeling markers by vascular cells and promoting the formation of a dense and disorganized collagen network. These findings identify scaffold-based shielding of cells from oscillatory shear stress as a potential handle to inhibit neointimal hyperplasia in grafts.

Inconsistency in Graft Outcome of Bilayered Bioresorbable Supramolecular Arterial Scaffolds in Rats.

There is a continuous search for the ideal bioresorbable material to develop scaffolds for in situ vascular tissue engineering. As these scaffolds are exposed to the harsh hemodynamic environment during the entire transformation process from scaffold to neotissue, it is of crucial importance to maintain mechanical integrity and stability at all times. Bilayered scaffolds made of supramolecular polycarbonate-ester-bisurea were manufactured using dual electrospinning. These scaffolds contained a porous inner layer to allow for cellular infiltration and a dense outer layer to provide strength. Scaffolds (n = 21) were implanted as an interposition graft into the abdominal aorta of male Lewis rats and explanted after 1, 3, and 5 months in vivo to assess mechanical functionality and neotissue formation upon scaffold resorption. Results demonstrated conflicting graft outcomes despite homogeneity in the experimental group and scaffold production. Most grafts exhibited adverse remodeling, resulting in aneurysmal dilatation and calcification. However, a few grafts did not demonstrate such features, but instead were characterized by graft extension and smooth muscle cell proliferation in the absence of endothelium, while remaining patent throughout the study. We conclude that it remains extremely difficult to anticipate graft development and performance in vivo. Next to rational mechanical design and good performance in vitro, a thorough understanding of the mechanobiological mechanisms governing scaffold-driven arterial regeneration as well as potential influences of surgical procedures is warranted to further optimize scaffold designs. Careful analysis of the differences between preclinical successes and failures, as is done in this study, may provide initial handles for scaffold optimization and standardized surgical procedures to improve graft performance in vivo. Impact statement In situ vascular tissue engineering using cell-free bioresorbable scaffolds is investigated as an off-the-shelf option to grow small caliber arteries inside the body. In this study, we developed a bilayered electrospun supramolecular scaffold with a dense outer layer to provide mechanical integrity and a porous inner layer for cell recruitment and tissue formation. Despite homogenous scaffold properties and mechanical performance in vitro, in vivo testing as rat aorta interposition grafts revealed distinct graft outcomes, ranging from aneurysms to functional arteries. Careful analysis of this variability provided valuable insights into materials-driven in situ artery formation relevant for scaffold design and implantation procedures.

Impact of Additives on Mechanical Properties of Supramolecular Electrospun Scaffolds.

The mechanical properties of scaffolds used for mechanically challenging applications such as cardiovascular implants are unequivocally important. Here, the effect of supramolecular additive functionalization on mechanical behavior of electrospun scaffolds was investigated for one bisurea-based model additive and two previously developed antifouling additives. The model additive has no effect on the mechanical properties of the bulk material, whereas the stiffness of electrospun scaffolds was slightly decreased compared to pristine PCL-BU following the addition of the three different additives. These results show the robustness of supramolecular additives used in biomedical applications, in which mechanical properties are important, such as vascular grafts and heart valve constructs.

Human In Vitro Model Mimicking Material-Driven Vascular Regeneration Reveals How Cyclic Stretch and Shear Stress Differentially Modulate Inflammation and Matrix Deposition.

Resorbable synthetic scaffolds designed to regenerate living tissues and organs inside the body have emerged as a clinically attractive technology to replace diseased blood vessels. However, mismatches between scaffold design and in vivo hemodynamic loading (i.e., cyclic stretch and shear stress) can result in aberrant inflammation and adverse tissue remodeling, leading to premature graft failure. Yet, the underlying mechanisms remain elusive. Here, a human in vitro model is presented that mimics the transient local inflammatory and biomechanical environments that drive scaffold-guided tissue regeneration. The model is based on the coculture of human (myo)fibroblasts and macrophages in a bioreactor platform that decouples cyclic stretch and shear stress. Using a resorbable supramolecular elastomer as the scaffold material, it is revealed that cyclic stretch initially reduces proinflammatory cytokine secretion and, especially when combined with shear stress, stimulates IL-10 secretion. Moreover, cyclic stretch stimulates downstream (myo)fibroblast proliferation and matrix deposition. In turn, shear stress attenuates cyclic-stretch-induced matrix growth by enhancing MMP-1/TIMP-1-mediated collagen remodeling, and synergistically alters (myo)fibroblast phenotype when combined with cyclic stretch. The findings suggest that shear stress acts as a stabilizing factor in cyclic stretch-induced tissue formation and highlight the distinct roles of hemodynamic loads in the design of resorbable vascular grafts.

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach-the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis-are discussed in detail.

Macrophage-Driven Biomaterial Degradation Depends on Scaffold Microarchitecture.

In situ tissue engineering is a technology in which non-cellular biomaterial scaffolds are implanted in order to induce local regeneration of replaced or damaged tissues. Degradable synthetic electrospun scaffolds are a versatile and promising class of biomaterials for various in situ tissue engineering applications, such as cardiovascular replacements. Functional in situ tissue regeneration depends on the balance between endogenous neo-tissue formation and scaffold degradation. Both these processes are driven by macrophages. Upon invasion into a scaffold, macrophages secrete reactive oxygen species (ROS) and hydrolytic enzymes, contributing to oxidative and enzymatic biomaterial degradation, respectively. This study aims to elucidate the effect of scaffold microarchitecture, i.e., µm-range fiber diameter and fiber alignment, on early macrophage-driven scaffold degradation. Electrospun poly-ε-caprolactone-bisurea (PCL-BU) scaffolds with either 2 or 6 µm (Ø) isotropic or anisotropic fibers were seeded with THP-1 derived human macrophages and cultured in vitro for 4 or 8 days. Our results revealed that macroph age-induced oxidative degradation in particular was dependent on scaffold microarchitecture, with the highest level of ROS-induced lipid peroxidation, NADPH oxidase gene expression and degradation in the 6 µm Ø anisotropic group. Whereas, biochemically polarized macrophages demonstrated a phenotype-specific degradative potential, the observed differences in macrophage degradative potential instigated by the scaffold microarchitecture could not be attributed to either distinct M1 or M2 polarization. This suggests that the scaffold microarchitecture uniquely affects macrophage-driven degradation. These findings emphasize the importance of considering the scaffold microarchitecture in the design of scaffolds for in situ tissue engineering applications and the tailoring of degradation kinetics thereof.

Hemodynamic loads distinctively impact the secretory profile of biomaterial-activated macrophages - implications for in situ vascular tissue engineering.

Biomaterials are increasingly used for in situ vascular tissue engineering, wherein resorbable fibrous scaffolds are implanted as temporary carriers to locally initiate vascular regeneration. Upon implantation, macrophages infiltrate and start degrading the scaffold, while simultaneously driving a healing cascade via the secretion of paracrine factors that direct the behavior of tissue-producing cells. This balance between neotissue formation and scaffold degradation must be maintained at all times to ensure graft functionality. However, the grafts are continuously exposed to hemodynamic loads, which can influence macrophage response in a hitherto unknown manner and thereby tilt this delicate balance. Here we aimed to unravel the effects of physiological levels of shear stress and cyclic stretch on biomaterial-activated macrophages, in terms of polarization, scaffold degradation and paracrine signaling to tissue-producing cells (i.e. (myo)fibroblasts). Human THP-1-derived macrophages were seeded in electrospun polycaprolactone bis-urea scaffolds and exposed to shear stress (∼1 Pa), cyclic stretch (∼1.04), or a combination thereof for 8 days. The results showed that macrophage polarization distinctly depended on the specific loading regime applied. In particular, hemodynamic loading decreased macrophage degradative activity, especially in conditions of cyclic stretch. Macrophage activation was enhanced upon exposure to shear stress, as evidenced from the upregulation of both pro- and anti-inflammatory cytokines. Exposure to the supernatant of these dynamically cultured macrophages was found to amplify the expression of tissue formation- and remodeling-related genes in (myo)fibroblasts statically cultured in comparable electrospun scaffolds. These results emphasize the importance of macrophage mechano-responsiveness in biomaterial-driven vascular regeneration.

Layer-specific cell differentiation in bi-layered vascular grafts under flow perfusion.

Bioengineered grafts have the potential to overcome the limitations of autologous and non-resorbable synthetic vessels as vascular substitutes. However, one of the challenges in creating these living grafts is to induce and maintain multiple cell phenotypes with a biomimetic organization. Our biomimetic grafts with heterotypic design hold promises for functional neovessel regeneration by guiding the layered cellular and tissue organization into a native-like structure. In this study, a perfusable two-compartment bioreactor chamber was designed for the further maturation of these vascular grafts, with a compartmentalized exposure of the graft's luminal and outer layer to cell-specific media. We used the system for a co-culture of endothelial colony forming cells and multipotent mesenchymal stromal cells (MSCs) in the vascular grafts, produced by combining electrospinning and melt electrowriting. It was demonstrated that the targeted cell phenotypes (i.e. endothelial cells (ECs) and vascular smooth muscle cells (vSMCs), respectively) could be induced and maintained during flow perfusion. The confluent luminal layer of ECs showed flow responsiveness, as indicated by the upregulation of COX-2, KLF2, and eNOS, as well as through stress fiber remodeling and cell elongation. In the outer layer, the circumferentially oriented, multi-layered structure of MSCs could be successfully differentiated into vSM-like cells using TGFß, as indicated by the upregulation of αSMA, calponin, collagen IV, and (tropo)elastin, without affecting the endothelial monolayer. The cellular layers inhibited diffusion between the outer and the inner medium reservoirs. This implies tightly sealed cellular layers in the constructs, resulting in truly separated bioreactor compartments, ensuring the exposure of the inner endothelium and the outer smooth muscle-like layer to cell-specific media. In conclusion, using this system, we successfully induced layer-specific cell differentiation with a native-like cell organization. This co-culture system enables the creation of biomimetic neovessels, and as such can be exploited to investigate and improve bioengineered vascular grafts.

Decoupling the Effect of Shear Stress and Stretch on Tissue Growth and Remodeling in a Vascular Graft.

The success of cardiovascular tissue engineering (TE) strategies largely depends on the mechanical environment in which cells develop a neotissue through growth and remodeling processes. This mechanical environment is defined by the local scaffold architecture to which cells adhere, that is, the microenvironment, and by external mechanical cues to which cells respond, that is, hemodynamic loading. The hemodynamic environment of early developing blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). Experimental platforms that recapitulate this mechanical environment in a controlled and tunable manner are thus critical for investigating cardiovascular TE. In traditional perfusion bioreactors, however, shear stress and stretch are coupled, hampering a clear delineation of their effects on cell and tissue response. In this study, we uniquely designed a bioreactor that independently combines these two types of mechanical cues in eight parallel vascular grafts. The system is computationally and experimentally validated, through finite element analysis and culture of tissue constructs, respectively, to distinguish various levels of shear stress (up to 5 Pa) and cyclic stretch (up to 1.10). To illustrate the usefulness of the system, we investigated the relative contribution of cyclic stretch (1.05 at 0.5 Hz) and shear stress (1 Pa) to tissue development. Both types of hemodynamic loading contributed to cell alignment, but the contribution of shear stress overruled stretch-induced cell proliferation and matrix (i.e., collagen and glycosaminoglycan) production. At a macroscopic level, cyclic stretching led to the most linear stress-stretch response, which was not related to the presence of shear stress. In conclusion, we have developed a bioreactor that is particularly suited to further unravel the interplay between hemodynamics and in situ TE processes. Using the new system, this work highlights the importance of hemodynamic loading to the study of developing vascular tissues.

Vascular Mechanobiology: Towards Control of In Situ Regeneration.

The paradigm of regenerative medicine has recently shifted from in vitro to in situ tissue engineering: implanting a cell-free, biodegradable, off-the-shelf available scaffold and inducing the development of functional tissue by utilizing the regenerative potential of the body itself. This approach offers a prospect of not only alleviating the clinical demand for autologous vessels but also circumventing the current challenges with synthetic grafts. In order to move towards a hypothesis-driven engineering approach, we review three crucial aspects that need to be taken into account when regenerating vessels: (1) the structure-function relation for attaining mechanical homeostasis of vascular tissues, (2) the environmental cues governing cell function, and (3) the available experimental platforms to test instructive scaffolds for in situ tissue engineering. The understanding of cellular responses to environmental cues leads to the development of computational models to predict tissue formation and maturation, which are validated using experimental platforms recapitulating the (patho)physiological micro-environment. With the current advances, a progressive shift is anticipated towards a rational and effective approach of building instructive scaffolds for in situ vascular tissue regeneration.

The initial repair response of articular cartilage after mechanically induced damage.

The regenerative potential of articular cartilage (AC) defects is limited and depends on defect size, biomechanical conditions, and age. Early events after overloading might be predictive for cartilage degeneration in the long term. Therefore, the present aim is to investigate the temporal response of cartilage to overloading at cell, matrix, and tissue level during the first period after mechanical overloading. In the present study, the effect of high loading (∼8 MPa) at a high rate (∼14 MPa/s) at day 0 during a 9 day period on collagen damage, gene expression, cell death, and biochemical composition in AC was investigated. A model system was developed which enabled culturing osteochondral explants after loading. Proteoglycan content was repeatedly monitored over time using µCT, whereas other evaluations required destructive measurements. Changes in matrix related gene expressions indicated a degenerative response during the first 6 h after loading. After 24 h, this was restored and data suggested an initial repair response. Cell death and microscopic damage increased after 24 h following loading. These degradative changes were not restored within the 9 day culture period, and were accompanied by a slight loss of proteoglycans at the articular surface that extended into the middle zones. The combined findings indicate that high magnitude loading of articular cartilage at a high rate induces an initial damage that later initiates a healing response that can probably not be retained due to loss of cell viability. Consequently, the matrix cannot be restored in the short term. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1265-1273, 2017.

On the importance of 3D, geometrically accurate, and subject-specific finite element analysis for evaluation of in-vivo soft tissue loads.

Pressure ulcers are a type of local soft tissue injury due to sustained mechanical loading and remain a common issue in patient care. People with spinal cord injury (SCI) are especially at risk of pressure ulcers due to impaired mobility and sensory perception. The development of load improving support structures relies on realistic tissue load evaluation e.g. using finite element analysis (FEA). FEA requires realistic subject-specific mechanical properties and geometries. This study focuses on the effect of geometry. MRI is used for the creation of geometrically accurate models of the human buttock for three able-bodied volunteers and three volunteers with SCI. The effect of geometry on observed internal tissue deformations for each subject is studied by comparing FEA findings for equivalent loading conditions. The large variations found between subjects confirms the importance of subject-specific FEA.