Shear flow affects selective monocyte recruitment into MCP-1-loaded scaffolds.

Novel cardiovascular replacements are being developed by using degradable synthetic scaffolds, which function as a temporary guide to induce neotissue formation directly in situ. Priming of such scaffolds with fast-releasing monocyte chemoattractant protein-1 (MCP-1) was shown to improve the formation of functional neoarteries in rats. However, the underlying mechanism has not been clarified. Therefore, the goal of this study was to investigate the effect of a burst-release of MCP-1 from a synthetic scaffold on the local recruitment of circulating leucocytes under haemodynamic conditions. Herein, we hypothesized that MCP-1 initiates a desired healing cascade by recruiting favourable monocyte subpopulations into the implanted scaffold. Electrospun poly(ε-caprolactone) scaffolds were loaded with fibrin gel containing various doses of MCP-1 and exposed to a suspension of human peripheral blood mononuclear cells in static or dynamic conditions. In standard migration assay, a dose-dependent migration of specific CD14(+) monocyte subsets was observed, as measured by flow cytometry. In conditions of pulsatile flow, on the other hand, a marked increase in immediate monocyte recruitment was observed, but without evident selectivity in monocyte subsets. This suggests that the selectivity was dependent on the release kinetics of the MCP-1, as it was overruled by the effect of shear stress after the initial burst-release. Furthermore, these findings demonstrate that local recruitment of specific MCP-1-responsive monocytes is not the fundamental principle behind the improved neotissue formation observed in long-term in vivo studies, and mobilization of MCP-1-responsive cells from the bone marrow into the bloodstream is suggested to play a predominant role in vivo.

Differential response of endothelial and endothelial colony forming cells on electrospun scaffolds with distinct microfiber diameters.

Electrospun scaffolds for in situ tissue engineering can be prepared with different fiber diameters to influence cell recruitment, adhesion, and differentiation. For cardiovascular applications, we investigated the impact of different fiber diameters (2, 5, 8, and 11 µm) in electrospun poly(ε-caprolactone) scaffolds on endothelial colony forming cells (ECFCs) in comparison to mature endothelial cells (HUVECs). In 2D cultures and on 2 µm fiber scaffolds, ECFC morphology and phenotype resemble those of HUVECs. When cultured on scaffolds with 5-11 µm fibers, a different behavior was detected. HUVECs developed a cytoskeleton organized circumferentially around the fibers, with collagen alignment in the same direction. ECFCs, instead, aligned the cytoskeleton along the scaffold fiber axis and deposited a homogeneous layer of collagen over the fibers; moreover, a subpopulation of ECFCs gained the αSMA marker. These results showed that ECFCs do not behave like mature endothelial cells in a 3D fibrous environment.

Functional regeneration at the blood-biomaterial interface.

The use of cardiovascular implants is commonplace in clinical practice. However, reproducing the key bioactive and adaptive properties of native cardiovascular tissues with an artificial replacement is highly challenging. Exciting new treatment strategies are under development to regenerate (parts of) cardiovascular tissues directly in situ using immunomodulatory biomaterials. Direct exposure to the bloodstream and hemodynamic loads is a particular challenge, given the risk of thrombosis and adverse remodeling that it brings. However, the blood is also a source of (immune) cells and proteins that dominantly contribute to functional tissue regeneration. This review explores the potential of the blood as a source for the complete or partial in situ regeneration of cardiovascular tissues, with a particular focus on the endothelium, being the natural blood-tissue barrier. We pinpoint the current scientific challenges to enable rational engineering and testing of blood-contacting implants to leverage the regenerative potential of the blood.

Donor Heterogeneity in the Human Macrophage Response to a Biomaterial Under Hyperglycemia In Vitro.

Macrophages have a commanding role in scaffold-driven in situ tissue regeneration. Depending on their polarization state, macrophages mediate the formation and remodeling of new tissue by secreting growth factors and cytokines. Therefore, successful outcomes of material-driven in situ tissue vascular tissue engineering depend largely on the immuno-regenerative potential of the recipient. A large cohort of patients requiring vascular replacements suffers from systemic multifactorial diseases, such as diabetes, which gives rise to a hyperglycemic and aggressive oxidative inflammatory environment that is hypothesized to hamper a well-balanced regenerative process. Here, we aimed at fundamentally exploring the effects of hyperglycemia, as one of the hallmarks of diabetes, on the macrophage response to three-dimensional (3D) electrospun synthetic biomaterials for in situ tissue engineering, in terms of inflammatory profile and tissue regenerative capacity. To simulate the early phases of the in situ regenerative cascade, we used a bottom-up in vitro approach. Primary human macrophages (n = 8 donors) were seeded in two-dimensional (2D) culture wells and polarized to pro-inflammatory M1 and anti-inflammatory M2 phenotype in normoglycemic (5.5 mM glucose), hyperglycemic (25 mM), and osmotic control (OC) conditions (5.5 mM glucose, 19.5 mM mannitol). Unpolarized macrophages and (myo)fibroblasts were seeded in mono- or co-culture in a 3D electrospun resorbable polycaprolactone bisurea scaffold and exposed to normoglycemic, hyperglycemic, and OC conditions. The results showed that macrophage polarization by biochemical stimuli was effective under all glycemic conditions and that the polarization states dictated expression of the receptors SCL2A1 (glucose transporter 1) and CD36 (fatty acid transporter). In 3D, the macrophage response to hyperglycemic conditions was strongly donor-dependent in terms of phenotype, cytokine secretion profile, and metabolic receptor expression. When co-cultured with (myo)fibroblasts, hyperglycemic conditions led to an increased expression of fibrogenic markers (ACTA2, COL1, COL3, IL-1ß). Together, these findings show that the hyperglycemic and hyperosmotic conditions may, indeed, influence the process of macrophage-driven in situ tissue engineering, and that the extent of this is likely to be patient-specific. Impact Statement Success or failure of cell-free bioresorbable in situ tissue-engineered vascular grafts hinges around the immuno-regenerative response of the recipient. Most patients requiring blood vessel replacements suffer from additional multifactorial diseases, such as diabetes, which may compromise their intrinsic regenerative potential. In this study, we used a bottom-up approach to study the effects of hyperglycemia, a hallmark of diabetes, on important phases in the in situ regenerative cascade, such as macrophage polarization and macrophage-myofibroblast crosstalk. The results demonstrate a relatively large donor-to-donor variation, which stresses the importance of taking scaffold-independent patient-specific factors into account when studying in situ biomaterial-driven tissue engineering.

Imparting Immunomodulatory Activity to Scaffolds via Biotin-Avidin Interactions.

Biotin-avidin interactions have been explored for decades as a technique to functionalize biomaterials, as well as for in vivo targeting, but whether changes in these interactions can be leveraged for immunomodulation remain unknown. The goal of this study was to investigate how biotin density and avidin variant can be used to deliver the immunomodulatory cytokine, interleukin 4 (IL4), from a porous gelatin scaffold, Gelfoam, to primary human macrophages in vitro. Here, we demonstrate that the degree of scaffold biotinylation controlled the binding of two different avidin variants, streptavidin and CaptAvidin. Biotinylated scaffolds were also loaded with streptavidin and biotinylated IL4 under flow, suggesting a potential use for targeting this biomaterial in vivo. While biotin-avidin interactions did not appear to influence the protein release in this system, increasing degrees of biotinylation did lead to increased M2-like polarization of primary human macrophages over time in vitro, highlighting the capability to leverage biotin-avidin interactions to modulate the macrophage phenotype. These results demonstrate a versatile and modular strategy to impart immunomodulatory activity to biomaterials.

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.

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.

In situ heart valve tissue engineering using a bioresorbable elastomeric implant - From material design to 12 months follow-up in sheep.

The creation of a living heart valve is a much-wanted alternative for current valve prostheses that suffer from limited durability and thromboembolic complications. Current strategies to create such valves, however, require the use of cells for in vitro culture, or decellularized human- or animal-derived donor tissue for in situ engineering. Here, we propose and demonstrate proof-of-concept of in situ heart valve tissue engineering using a synthetic approach, in which a cell-free, slow degrading elastomeric valvular implant is populated by endogenous cells to form new valvular tissue inside the heart. We designed a fibrous valvular scaffold, fabricated from a novel supramolecular elastomer, that enables endogenous cells to enter and produce matrix. Orthotopic implantations as pulmonary valve in sheep demonstrated sustained functionality up to 12 months, while the implant was gradually replaced by a layered collagen and elastic matrix in pace with cell-driven polymer resorption. Our results offer new perspectives for endogenous heart valve replacement starting from a readily-available synthetic graft that is compatible with surgical and transcatheter implantation procedures.

Development of Non-Cell Adhesive Vascular Grafts Using Supramolecular Building Blocks.

Cell-free approaches to in situ tissue engineering require materials that are mechanically stable and are able to control cell-adhesive behavior upon implantation. Here, the development of mechanically stable grafts with non-cell adhesive properties via a mix-and-match approach using ureido-pyrimidinone (UPy)-modified supramolecular polymers is reported. Cell adhesion is prevented in vitro through mixing of end-functionalized or chain-extended UPy-polycaprolactone (UPy-PCL or CE-UPy-PCL, respectively) with end-functionalized UPy-poly(ethylene glycol) (UPy-PEG) at a ratio of 90:10. Further characterization reveals intimate mixing behavior of UPy-PCL with UPy-PEG, but poor mechanical properties, whereas CE-UPy-PCL scaffolds are mechanically stable. As a proof-of-concept for the use of non-cell adhesive supramolecular materials in vivo, electrospun vascular scaffolds are applied in an aortic interposition rat model, showing reduced cell infiltration in the presence of only 10% of UPy-PEG. Together, these results provide the first steps toward advanced supramolecular biomaterials for in situ vascular tissue engineering with control over selective cell capturing.

In Situ Tissue Engineering of Functional Small-Diameter Blood Vessels by Host Circulating Cells Only.

Inflammation is a natural phase of the wound healing response, which can be harnessed for the in situ tissue engineering of small-diameter blood vessels using instructive, bioresorbable synthetic grafts. This process is dependent on colonization of the graft by host circulating cells and subsequent matrix formation. Typically, vascular regeneration in small animals is governed by transanastomotic cell ingrowth. However, this process is very rare in humans and hence less relevant for clinical translation. Therefore, a novel rat model was developed, in which cell ingrowth from the adjacent tissue is inhibited using Gore-Tex sheathing. Using this model, our aim here was to prove that functional blood vessels can be formed in situ through the host inflammatory response, specifically by blood-borne cells. The model was validated by implanting sex-mismatched aortic segments on either anastomoses of an electrospun poly(ɛ-caprolactone) (PCL) graft, filled with fibrin gel, into the rat abdominal aorta. Fluorescent in situ hybridization analysis revealed that after 1 and 3 months in vivo, over 90% of infiltrating cells originated from the bloodstream, confirming the effective shielding of transanastomotic cell ingrowth. Using the validated model, PCL/fibrin grafts were implanted, either or not loaded with monocyte chemotactic protein-1 (MCP-1), and cell infiltration and tissue development were investigated at various key time points in the healing cascade. A phased healing response was observed, initiated by a rapid influx of inflammatory cells, mediated by the local release of MCP-1. After 3 months in vivo, the grafts consisted of a medial layer with smooth muscle cells in an oriented collagen matrix, an intimal layer with elastin fibers, and confluent endothelium. This study proves the regenerative potential of cells in the circulatory system in the setting of in situ vascular tissue engineering.

Synergistic protein secretion by mesenchymal stromal cells seeded in 3D scaffolds and circulating leukocytes in physiological flow.

Mesenchymal stromal cells (MSC) play an important role in natural wound healing via paracrine and juxtacrine signaling to immune cells. The aim of this study was to identify the signaling factors secreted by preseeded cells in a biomaterial and their interaction with circulating leukocytes, in the presence of physiological biomechanical stimuli exerted by the hemodynamic environment (i.e. strain and shear flow). Electrospun poly(ε-caprolactone)-based scaffolds were seeded with human peripheral blood mononuclear cells (PBMC) or MSC. Protein secretion was analyzed under static conditions and cyclic strain. Subsequently, the cross-talk between preseeded cells and circulating leukocytes was addressed by exposing the scaffolds to a suspension of PBMC in static transwells and in pulsatile flow. Our results revealed that PBMC exposed to the scaffold consistently secreted a cocktail of immunomodulatory proteins under all conditions tested. Preseeded MSC, on the other hand, secreted the trophic factors MCP-1, VEGF and bFGF. Furthermore, we observed a synergistic upregulation of CXCL12 gene expression and a synergistic increase in bFGF protein production by preseeded MSC exposed to PBMC in pulsatile flow. These findings identify CXCL12 and bFGF as valuable targets for the development of safe and effective acellular instructive grafts for application in in situ cardiovascular regenerative therapies.

A mesofluidics-based test platform for systematic development of scaffolds for in situ cardiovascular tissue engineering.

Recently, in situ tissue engineering has emerged as a new approach to obtain autologous, living replacement tissues with off-the-shelf availability. The method is based on the use of an instructive biodegradable scaffold that is capable of repopulation with host cells in situ and subsequent tissue formation. This approach imposes high demands on scaffold properties. For cardiovascular grafts, the repopulation with endogenous cells from the circulation is further hypothesized to be influenced by the hemodynamic environment of the scaffold. To systematically study the effect of scaffold properties on the response of circulating cells, we aimed to develop a mesofluidics-based in vitro test platform that enables on-stage investigation of the interaction of circulating cells with three-dimensional (3D) synthetic scaffolds under physiologic hemodynamic conditions. The test platform consists of a custom-developed cross-flow chamber that houses small-scale 3D scaffolds. The cross-flow chamber is incorporated into a flow-loop to drive a cell suspension along the scaffold with physiological wall shear stress and perfusion pressure. The fluidics system is validated numerically and experimentally using a computational fluid dynamics model and real-time microbead tracing studies, demonstrating a fully developed flow profile with a homogeneous shear stress distribution over the scaffold. Wall shear stresses and pressure can be controlled independently, well within the target physiological range (0-8 Pa and 0-100 mmHg, respectively). Bench-top evaluation is performed using electrospun poly(ɛ-caprolactone) scaffolds with varying fiber diameter, exposed to a suspension of human peripheral blood mononuclear cells in pulsatile flow for 72 h. Cell adhesion and infiltration are monitored using time-lapsed confocal laser scanning microscopy. In conclusion, we have successfully developed a mesofluidics platform to study cell-scaffold interactions under hemodynamic conditions in vitro. This platform not only enables us to systematically screen and develop potential scaffolds for future in situ cardiovascular tissue engineering approaches, but also acts as a tool to further elucidate processes as observed in vivo.