Modulation of macrophage phenotype and protein secretion via heparin-IL-4 functionalized supramolecular elastomers.

Hallmark of the in situ tissue engineering approach is the use of bioresorbable, synthetic, acellular scaffolds, which are designed to modulate the inflammatory response and actively trigger tissue regeneration by the body itself at the site of implantation. Much research is devoted to the design of synthetic materials modulating the polarization of macrophages, which are essential mediators of the early stages of the inflammatory response. Here, we present a novel method for the functionalization of elastomers based on synthetic peptide chemistry, supramolecular self-assembly, and immobilization of heparin and interleukin 4 (IL-4), which is known to skew the polarization of macrophages into the wound healing "M2" phenotype. Ureido-pyrimidinone (UPy)-modified chain extended polycaprolactone (CE-UPy-PCL) was mixed with a UPy-modified heparin binding peptide (UPy-HBP) to allow for immobilization of heparin, and further functionalization with IL-4 via its heparin binding domain. As a first proof of principle, CE-UPy-PCL and UPy-HBP were premixed in solution, dropcast and exposed to primary human monocyte-derived macrophages, in the presence or absence of IL-4-heparin functionalization. It was demonstrated that the supramolecular IL-4-heparin functionalization effectively promoted macrophage polarization into an anti-inflammatory phenotype, in terms of morphology, immunohistochemistry and cytokine secretion. Moreover, the supramolecular functionalization approach used was successfully translated to 3D electrospun scaffolds for in situ tissue engineering purposes, where UPy-HBP retention, and heparin and IL-4 attachment to the supramolecular scaffolds were proven over 7 days. Lastly, human monocyte-derived macrophages were cultured on 3D scaffolds, which, in case of IL-4-heparin functionalization, were proven to promote of an anti-inflammatory environment on protein level. This study presents a novel method in designing a versatile class of functionalized elastomers that effectively harness the anti-inflammatory behavior of macrophages in vitro, and as such, may be instrumental for the development of a new class of synthetic materials for in situ tissue engineering purposes. STATEMENT OF SIGNIFICANCE: Macrophages and their phenotypic and functional plasticity play a pivotal role in metabolic homeostasis and tissue repair. Based on this notion, bioactivated materials modulating macrophage polarization were extensively investigated in the past. Here, we designed immunomodulating, synthetic materials based on supramolecular immobilization of a heparin binding peptide, and further bioactivation with heparin and IL-4, an anti-inflammatory cytokine responsible for M2 activation and polarization. Human monocyte-derived macrophages cultured on heparin-IL-4 bioactivated materials displayed an elongated morphology and an anti-inflammatory phenotype, with downregulation of pro-inflammatory cytokines and promotion of anti-inflammatory cytokines over time. This study represents the first step in designing a novel class of synthetic, bioactivated materials that harness the regenerative behavior of host macrophages towards in situ tissue regeneration.

Modulation of collagen fiber orientation by strain-controlled enzymatic degradation.

Collagen fiber anisotropy has a significant influence on the function and mechanical properties of cardiovascular tissues. We investigated if strain-dependent collagen degradation can explain collagen orientation in response to uniaxial and biaxial mechanical loads. First, decellularized pericardial samples were stretched to a fixed uniaxial strain and after adding a collagen degrading enzyme (collagenase), force relaxation was measured to calculate the degradation rate. This data was used to identify the strain-dependent degradation rate. A minimum was observed in the degradation rate curve. It was then demonstrated, for the first time, that biaxial strain in combination with collagenase alters the collagen fiber alignment from an initially isotropic distribution to an anisotropic distribution with a mean alignment corresponding with the strain at the minimum degradation rate, which may be in between the principal strain directions. When both strains were smaller than the minimum degradation point, fibers tended to align in the direction of the larger strain and when both strains were larger than the minimum degradation, fibers mainly aligned in the direction of the smaller strain. However, when one strain was larger and one was smaller than the minimum degradation point, the observed fiber alignment was in between the principal strain directions. In the absence of collagenase, uniaxial and biaxial strains only had a slight effect on the collagen (re)orientation of the decellularized samples. STATEMENT OF SIGNIFICANCE: Collagen fiber orientation is a significant determinant of the mechanical properties of native tissues. To mimic the native-like collagen alignment in vitro, we need to understand the underlying mechanisms that direct this alignment. In the current study, we aimed to control collagen fiber orientation by applying biaxial strains in the presence of collagenase. We hypothesized that strain-dependent collagen degradation can describe specific collagen orientation when biaxial mechanical strains are applied. Based on this hypothesis, collagen fibers align in the direction where the degradation is minimal. Pericardial tissues, as isotropic collagen matrices, were decellularized and subjected to a fixed uniaxial strain. Then, collagenase was added to initiate the collagen degradation and the relaxation of force was measured to indicate the degradation rate. The V-shaped relationship between degradation rate and strain was obtained to identify the minimum degradation rate point. It was then demonstrated, for the first time, that biaxial strain in combination with collagenase alters the collagen fiber alignment from almost isotropic to a direction corresponding with the strain at the minimum degradation rate.

Hydrolytic and oxidative degradation of electrospun supramolecular biomaterials: In vitro degradation pathways.

The emerging field of in situ tissue engineering (TE) of load bearing tissues places high demands on the implanted scaffolds, as these scaffolds should provide mechanical stability immediately upon implantation. The new class of synthetic supramolecular biomaterial polymers, which contain non-covalent interactions between the polymer chains, thereby forming complex 3D structures by self assembly. Here, we have aimed to map the degradation characteristics of promising (supramolecular) materials, by using a combination of in vitro tests. The selected biomaterials were all polycaprolactones (PCLs), either conventional and unmodified PCL, or PCL with supramolecular hydrogen bonding moieties (either 2-ureido-[1H]-pyrimidin-4-one or bis-urea units) incorporated into the backbone. As these materials are elastomeric, they are suitable candidates for cardiovascular TE applications. Electrospun scaffold strips of these materials were incubated with solutions containing enzymes that catalyze hydrolysis, or solutions containing oxidative species. At several time points, chemical, morphological, and mechanical properties were investigated. It was demonstrated that conventional and supramolecular PCL-based polymers respond differently to enzyme-accelerated hydrolytic or oxidative degradation, depending on the morphological and chemical composition of the material. Conventional PCL is more prone to hydrolytic enzymatic degradation as compared to the investigated supramolecular materials, while, in contrast, the latter materials are more susceptible to oxidative degradation. Given the observed degradation pathways of the examined materials, we are able to tailor degradation characteristics by combining selected PCL backbones with additional supramolecular moieties. The presented combination of in vitro test methods can be employed to screen, limit, and select biomaterials for pre-clinical in vivo studies targeted to different clinical applications.

A comparative analysis of the collagen architecture in the carotid artery: second harmonic generation versus diffusion tensor imaging.

Collagen is the main load-bearing component of the artery. The 3D arrangement of the collagen fibers is crucial to understand the mechanical behavior of such tissues. We compared collagen fiber alignment obtained by second harmonic generation (SHG) microscopy with the alignment obtained by diffusion tensor imaging (DTI) throughout the wall of a porcine carotid artery to check the feasibility of using DTI as a fast and non-destructive method instead of SHG. The middle part of the artery was cut into two segments: one for DTI and one for the SHG measurements. The tissue for SHG measurements was cut into 30µm tangential sections. After scanning all sections, they were registered together and the fiber orientation was quantified by an in-house algorithm. The tissue for DTI measurement was embedded in type VII agarose and scanned with an MRI-scanner. Fiber tractography was performed on the DTI images. Both methods showed a layered structure of the wall. The fibers were mainly oriented circumferentially in the outer adventitia and media. DTI revealed the predominant layers of the arterial wall. This study showed the feasibility of using DTI for evaluating the collagen orientation in native artery as a fast and non-destructive method.

Substrates for cardiovascular tissue engineering.

Cardiovascular tissue engineering aims to find solutions for the suboptimal regeneration of heart valves, arteries and myocardium by creating 'living' tissue replacements outside (in vitro) or inside (in situ) the human body. A combination of cells, biomaterials and environmental cues of tissue development is employed to obtain tissues with targeted structure and functional properties that can survive and develop within the harsh hemodynamic environment of the cardiovascular system. This paper reviews the up-to-date status of cardiovascular tissue engineering with special emphasis on the development and use of biomaterial substrates. Key requirements and properties of these substrates, as well as methods and readout parameters to test their efficacy in the human body, are described in detail and discussed in the light of current trends toward designing biologically inspired microenviroments for in situ tissue engineering purposes.