Computation meets experiment: identification of highly efficient fibrillating peptides.

Self-assembling peptides are of huge interest for biological, medical and nanotechnological applications. The enormous chemical variety that is available from the 20 amino acids offers potentially unlimited peptide sequences, but it is currently an issue to predict their supramolecular behavior in a reliable and cheap way. Herein we report a computational method to screen and forecast the aqueous self-assembly propensity of amyloidogenic pentapeptides. This method was found also as an interesting tool to predict peptide crystallinity, which may be of interest for the development of peptide based drugs.

Emergence of Elastic Properties in a Minimalist Resilin-Derived Heptapeptide upon Bromination.

Bromination is herein exploited to promote the emergence of elastic behavior in a short peptide-SDSYGAP-derived from resilin, a rubber-like protein exerting its role in the jumping and flight systems of insects. Elastic and resilient hydrogels are obtained, which also show self-healing behavior, thanks to the promoted non-covalent interactions that limit deformations and contribute to the structural recovery of the peptide-based hydrogel. In particular, halogen bonds may stabilize the ß-sheet organization working as non-covalent cross-links between nearby peptide strands. Importantly, the unmodified peptide (i.e., wild type) does not show such properties. Thus, SDSY(3,5-Br)GAP is a novel minimalist peptide elastomer.

Positive impact of dynamic seeding of mesenchymal stem cells on bone-like biodegradable scaffolds with increased content of calcium phosphate nanoparticles.

One of the main aims of bone tissue engineering, regenerative medicine and cell therapy is development of an optimal artificial environment (scaffold) that can trigger a favorable response within the host tissue, it is well colonized by resident cells of organism and ideally, it can be in vitro pre-colonized by cells of interest to intensify the process of tissue regeneration. The aim of this study was to develop an effective tool for regenerative medicine, which combines the optimal bone-like scaffold and colonization technique suitable for cell application. Accordingly, this study includes material (physical, chemical and structural) and in vitro biological evaluation of scaffolds prior to in vivo study. Thus, porosity, permeability or elasticity of two types of bone-like scaffolds differing in the ratio of collagen type I and natural calcium phosphate nanoparticles (bCaP) were determined, then analyzes of scaffold interaction with mesenchymal stem cells (MSCs) were performed. Simultaneously, dynamic seeding using a perfusion bioreactor followed by static cultivation was compared with standard static cultivation for the whole period of cultivation. In summary, cell colonization ability was estimated by determination of cell distribution within the scaffold (number, depth and homogeneity), matrix metalloproteinase activity and gene expression analysis of signaling molecules and differentiation markers. Results showed, the used dynamic colonization technique together with the newly-developed collagen-based scaffold with high content of bCaP to be an effective combined tool for producing bone grafts for bone implantology and regenerative medicine.

Dry versus hydrated collagen scaffolds: are dry states representative of hydrated states?

Collagen composite scaffolds have been used for a number of studies in tissue engineering. The hydration of such highly porous and hydrophilic structures may influence mechanical behaviour and porosity due to swelling. The differences in physical properties following hydration would represent a significant limiting factor for the seeding, growth and differentiation of cells in vitro and the overall applicability of such hydrophilic materials in vivo. Scaffolds based on collagen matrix, poly(DL-lactide) nanofibers, calcium phosphate particles and sodium hyaluronate with 8 different material compositions were characterised in the dry and hydrated states using X-ray microcomputed tomography, compression tests, hydraulic permeability measurement, degradation tests and infrared spectrometry. Hydration, simulating the conditions of cell seeding and cultivation up to 48 h and 576 h, was found to exert a minor effect on the morphological parameters and permeability. Conversely, hydration had a major statistically significant effect on the mechanical behaviour of all the tested scaffolds. The elastic modulus and compressive strength of all the scaffolds decreased by ~95%. The quantitative results provided confirm the importance of analysing scaffolds in the hydrated rather than the dry state since the former more precisely simulates the real environment for which such materials are designed.

On-chip assessment of human primary cardiac fibroblasts proliferative responses to uniaxial cyclic mechanical strain.

Cardiac cell function is substantially influenced by the nature and intensity of the mechanical loads the cells experience. Cardiac fibroblasts (CFs) are primarily involved in myocardial tissue remodeling: at the onset of specific pathological conditions, CFs activate, proliferate, differentiate, and critically alter the amount of myocardial extra-cellular matrix with important consequences for myocardial functioning. While cyclic mechanical strain has been shown to increase matrix synthesis of CFs in vitro, the role of mechanical cues in CFs proliferation is unclear. We here developed a multi-chamber cell straining microdevice for cell cultures under uniform, uniaxial cyclic strain. After careful characterization of the strain field, we extracted human heart-derived CFs and performed cyclic strain experiments. We subjected cells to 2% or 8% cyclic strain for 24 h or 72 h, using immunofluorescence to investigate markers of cell morphology, cell proliferation (Ki67, EdU, phospho-Histone-H3) and subcellular localization of the mechanotransduction-associated transcription factor YAP. Cell morphology was affected by cyclic strain in terms of cell area, cell and nuclear shape and cellular alignment. We additionally observed a strain intensity-dependent control of cell growth: a significant proliferation increase occurred at 2% cyclic strain, while time-dependent effects took place upon 8% cyclic strain. The YAP-dependent mechano-transduction pathway was similarly activated in both strain conditions. These results demonstrate a differential effect of cyclic strain intensity on human CFs proliferation control and provide insights into the YAP-dependent mechano-sensing machinery of human CFs.

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.

3D photopolymerized microstructured scaffolds influence nuclear deformation, nucleo/cytoskeletal protein organization, and gene regulation in mesenchymal stem cells.

Mechanical stimuli from the extracellular environment affect cell morphology and functionality. Recently, we reported that mesenchymal stem cells (MSCs) grown in a custom-made 3D microscaffold, the Nichoid, are able to express higher levels of stemness markers. In fact, the Nichoid is an interesting device for autologous MSC expansion in clinical translation and would appear to regulate gene activity by altering intracellular force transmission. To corroborate this hypothesis, we investigated mechanotransduction-related nuclear mechanisms, and we also treated spread cells with a drug that destroys the actin cytoskeleton. We observed a roundish nuclear shape in MSCs cultured in the Nichoid and correlated the nuclear curvature with the import of transcription factors. We observed a more homogeneous euchromatin distribution in cells cultured in the Nichoid with respect to the Flat sample, corresponding to a standard glass coverslip. These results suggest a different gene regulation, which we confirmed by an RNA-seq analysis that revealed the dysregulation of 1843 genes. We also observed a low structured lamina mesh, which, according to the implemented molecular dynamic simulations, indicates reduced damping activity, thus supporting the hypothesis of low intracellular force transmission. Also, our investigations regarding lamin expression and spatial organization support the hypothesis that the gene dysregulation induced by the Nichoid is mainly related to a reduction in force transmission. In conclusion, our findings revealing the Nichoid's effects on MSC behavior is a step forward in the control of stem cells via mechanical manipulation, thus paving the way to new strategies for MSC translation to clinical applications.

Hybrid fibroin/polyurethane small-diameter vascular grafts: from fabrication toin vivopreliminary assessment.

To address the need of alternatives to autologous vessels for small-calibre vascular applications (e.g. cardiac surgery), a bio-hybrid semi-degradable material composed of silk fibroin (SF) and polyurethane (Silkothane®) was herein used to fabricate very small-calibre grafts (Øin= 1.5 mm) via electrospinning. Bio-hybrid grafts werein vitrocharacterized in terms of morphology and mechanical behaviour, and compared to similar grafts of pure SF. Similarly, two native vessels from a rodent model (abdominal aorta and vena cava) were harvested and characterized. Preliminary implants were performed on Lewis rats to confirm the suitability of Silkothane® grafts for small-calibre applications, specifically as aortic insertion and femoral shunt. The manufacturing process generated pliable grafts consisting of a randomized fibrous mesh and exhibiting similar geometrical features to rat aortas. Both Silkothane® and pure SF grafts showed radial compliances in the range from 1.37 ± 0.86 to 1.88 ± 1.01% 10-2mmHg-1, lower than that of native vessels. The Silkothane® small-calibre devices were also implanted in rats demonstrating to be adequate for vascular applications; all the treated rats survived the surgery for three months after implantation, and 16 rats out of 17 (94%) still showed blood flow inside the graft at sacrifice. The obtained results lay the basis for a deeper investigation of the interaction between the Silkothane® graft and the implant site, which may deal with further analysis on the potentialities in terms of degradability and tissue formation, on longer time-points.

Trends in biomedical engineering: focus on Regenerative Medicine.

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.

Anisotropic elastic network modeling of entire microtubules.

Microtubules are supramolecular structures that make up the cytoskeleton and strongly affect the mechanical properties of the cell. Within the cytoskeleton filaments, the microtubule (MT) exhibits by far the highest bending stiffness. Bending stiffness depends on the mechanical properties and intermolecular interactions of the tubulin dimers (the MT building blocks). Computational molecular modeling has the potential for obtaining quantitative insights into this area. However, to our knowledge, standard molecular modeling techniques, such as molecular dynamics (MD) and normal mode analysis (NMA), are not yet able to simulate large molecular structures like the MTs; in fact, their possibilities are normally limited to much smaller protein complexes. In this work, we developed a multiscale approach by merging the modeling contribution from MD and NMA. In particular, MD simulations were used to refine the molecular conformation and arrangement of the tubulin dimers inside the MT lattice. Subsequently, NMA was used to investigate the vibrational properties of MTs modeled as an elastic network. The coarse-grain model here developed can describe systems of hundreds of interacting tubulin monomers (corresponding to up to 1,000,000 atoms). In particular, we were able to simulate coarse-grain models of entire MTs, with lengths up to 350 nm. A quantitative mechanical investigation was performed; from the bending and stretching modes, we estimated MT macroscopic properties such as bending stiffness, Young modulus, and persistence length, thus allowing a direct comparison with experimental data.

Mechanical characterization of actomyosin complex by molecular mechanics simulations.

PURPOSE: Knowledge of the mechanical behavior of myosin and actin monomer is critical for understanding the molecular mechanism of actomyosin-based muscle and non-muscle motility. Different experimental studies concerning actomyosin interaction have been performed in vitro, but studies at the single molecule level have just begun. The aim of this study was to provide a mechanical characterization of myosin II and actin monomer using a numerical approach. METHODS: The elastic properties of the two proteins involved in muscle contraction were assessed by performing stretching simulations up to 10% protein elongation using the restraining method. Interaction properties of the actomyosin complex were evaluated at eight intermolecular distances during which the entire system was left free to move. RESULTS: According to our results, the values of the elastic modulus of the myosin motor domain and actin are 0.30 GPa, and 0.08 GPa, respectively. As for the actomyosin complex, the interaction force has a maximum value of 541.15 pN. CONCLUSIONS: Mechanical properties of molecular motors are currently being debated. Our results match a number of experimental data, therefore, supporting the idea that molecular mechanics may be a powerful tool to find a way in this complex subject.

Natural and Synthetic Polymers for Bone Scaffolds Optimization.

Bone tissue is the structural component of the body, which allows locomotion, protects vital internal organs, and provides the maintenance of mineral homeostasis. Several bone-related pathologies generate critical-size bone defects that our organism is not able to heal spontaneously and require a therapeutic action. Conventional therapies span from pharmacological to interventional methodologies, all of them characterized by several drawbacks. To circumvent these effects, tissue engineering and regenerative medicine are innovative and promising approaches that exploit the capability of bone progenitors, especially mesenchymal stem cells, to differentiate into functional bone cells. So far, several materials have been tested in order to guarantee the specific requirements for bone tissue regeneration, ranging from the material biocompatibility to the ideal 3D bone-like architectural structure. In this review, we analyse the state-of-the-art of the most widespread polymeric scaffold materials and their application in in vitro and in vivo models, in order to evaluate their usability in the field of bone tissue engineering. Here, we will present several adopted strategies in scaffold production, from the different combination of materials, to chemical factor inclusion, embedding of cells, and manufacturing technology improvement.

Advantages and limitations of a supernegative GFP in facilitating MyoD intracellular tracking.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (-30)GFP, to a myogenic transcription factor, MyoD. The (-30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (-30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (-30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

Coronary artery mechanics induces human saphenous vein remodelling via recruitment of adventitial myofibroblast-like cells mediated by Thrombospondin-1.

Rationale: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. Methods: We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results in vitro and using a model of SV carotid interposition in pigs. Results: Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-ß-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-ß-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. Conclusions: Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors.

Aortic root performance after valve sparing procedure: a comparative finite element analysis.

David and Yacoub sparing techniques are the most common procedures adopted for the surgical correction of aortic root aneurysms. These surgical procedures entail the replacement of the sinuses of Valsalva with a synthetic graft, inside which the cusps are re-suspended. Root replacement by a synthetic graft may result in altered valve behaviour both in terms of coaptation and stress distribution, thus leading to the failure of the correction. A finite element approach was used to investigate this phenomenon; four 3D models of the aortic root were developed to simulate the root in physiological, pathological and post-operative conditions after the two different surgical procedures. The physiological 3D geometrical model was developed on the basis of anatomical data obtained from echocardiographic images; it was then modified to obtain the pathological and post-operative models. The effectiveness of both techniques was assessed by comparison with the first two simulated conditions, in terms of stresses acting on the root, leaflet coaptation and interaction between leaflets and the graft during valve opening. Results show that both sparing techniques are able to restore aortic valve coaptation and to reduce stresses induced by the initial root dilation. Nonetheless, both techniques lead to altered leaflet kinematics, with more evident alterations after David repair.

Interaction forces and interface properties of KIF1A kinesin-alphabeta tubulin complex assessed by molecular dynamics.

Kinesin is a microtubule-based motor protein that generates motion involved in intracellular trafficking and cell division. Even if the force-generating and enzymatic properties of kinesin were extensively studied, the molecular basis of its interaction with the microtubule is still not well understood. The aim of the present study is to provide a detailed description, in terms of conformational changes and interaction properties, of the kinesin-alphabeta tubulin complex during a cycle of ATP hydrolysis. Four different nucleotide-dependent conformations (nucleotide-free, ATP, ADP.Pi and ADP) of the kinesin-alphabeta tubulin were constructed and investigated by performing molecular dynamics simulations. Computational results show that small conformational changes, in the order of few Angstrom, occurring in the kinesin structure reflect on its affinity for the filament substrate. Indeed the rotation of the alpha4 helix due to the transition from the bound (ADP.Pi) to the unbound (ADP) state, when the Pi is released from the complex, coupled with the modification occurred in the loop L9 of switch I domain are associated to a marked decrease (approximately 45%) of the maximum interaction force between the kinesin motor and the tubulin dimer.

Actomyosin interaction: mechanical and energetic properties in different nucleotide binding states.

The mechanics of the actomyosin interaction is central in muscle contraction and intracellular trafficking. A better understanding of the events occurring in the actomyosin complex requires the examination of all nucleotide-dependent states and of the energetic features associated with the dynamics of the cross-bridge cycle. The aim of the present study is to estimate the interaction strength between myosin in nucleotide-free, ATP, ADP.Pi and ADP states and actin monomer. The molecular models of the complexes were constructed based on cryo-electron microscopy maps and the interaction properties were estimated by means of a molecular dynamics approach, which simulate the unbinding of the complex applying a virtual spring to the core of myosin protein. Our results suggest that during an ATP hydrolysis cycle the affinity of myosin for actin is modulated by the presence and nature of the nucleotide in the active site of the myosin motor domain. When performing unbinding simulations with a pulling rate of 0.001 nm/ps, the maximum pulling force applied to the myosin during the experiment is about 1nN. Under these conditions the interaction force between myosin and actin monomer decreases from 0.83 nN in the nucleotide-free state to 0.27 nN in the ATP state, and increases to 0.60 nN after ATP hydrolysis and Pi release from the complex (ADP state).

Assembly of Tissue-Engineered Blood Vessels with Spatially Controlled Heterogeneities.

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue-engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of certain vascular diseases. We developed a system for generating self-assembled human smooth muscle cell (SMC) ring units, which were fused together into TEBVs. The goal of this study was to assess the feasibility of modular assembly and fusion of ring building units to fabricate spatially controlled, heterogeneous tissue tubes. We first aimed to enhance fusion and reduce total culture time, and determined that reducing ring preculture duration improved tube fusion. Next, we incorporated electrospun polymer ring units onto tube ends as reinforced extensions, which allowed us to cannulate tubes after only 7 days of fusion, and culture tubes with luminal flow in a custom bioreactor. To create focal heterogeneities, we incorporated gelatin microspheres into select ring units during self-assembly, and fused these rings between ring units without microspheres. Cells within rings maintained their spatial position along tissue tubes after fusion. Because tubes fabricated from primary SMCs did not express contractile proteins, we also fabricated tubes from human mesenchymal stem cells, which expressed smooth muscle alpha actin and SM22-α. This work describes a platform approach for creating modular TEBVs with spatially defined structural heterogeneities, which may ultimately be applied to mimic focal diseases such as intimal hyperplasia or aneurysm.

Engineering an Environment for the Study of Fibrosis: A 3D Human Muscle Model with Endothelium Specificity and Endomysium.

The integration of vascular structures into in vitro cultured tissues provides realistic models of complex tissue-vascular interactions. Despite the incidence and impact of muscle-wasting disorders, advanced in vitro systems are still far from recapitulating the environmental complexity of skeletal muscle. Our model comprises differentiated human muscle fibers enveloped by a sheath of human muscle-derived fibroblasts and supported by a vascular network with mural-like cells. Here, we demonstrate the induction of muscle-specific endothelium and the self-organization of endomysial muscle fibroblasts mediated by endothelial cells. We use this model to mimic the fibrotic environment characterizing muscular dystrophies and to highlight key signatures of fibrosis that are neglected or underestimated in traditional 2D monocultures. Overall, this vascularized meso-scale cellular construct finely recapitulates the human skeletal muscle environment and provides an advanced solution for in vitro studies of muscle physiology and pathology.

Versican is differentially regulated in the adventitial and medial layers of human vein grafts.

Changes in extracellular matrix proteins may contribute significantly to the adaptation of vein grafts to the arterial circulation. We examined the production and distribution of versican and hyaluronan in intact human vein rings cultured ex vivo, veins perfused ex vivo, and cultured venous adventitial and smooth muscle cells. Immunohistochemistry revealed higher levels of versican in the intima/media compared to the adventitia, and no differences in hyaluronan. In the vasa vasorum, versican and hyaluronan associated with CD34+ progenitor cells. Culturing the vein rings for 14 days revealed increased versican immunostaining of 30-40% in all layers, with no changes in hyaluronan. Changes in versican accumulation appear to result from increased synthesis in the intima/media and decreased degradation in the adventitia as versican transcripts were increased in the intima/media, but unchanged in the adventitia, and versikine (the ADAMTS-mediated cleavage product of versican) was increased in the intima/media, but decreased in the adventitia. In perfused human veins, versican was specifically increased in the intima/media in the presence of venous pressure, but not with arterial pressure. Unexpectedly, cultured adventitial cells express and accumulate more versican and hyaluronan than smooth muscle cells. These data demonstrate a differential regulation of versican and hyaluronan in human venous adventitia vs. intima/media and suggest distinct functions for these extracellular matrix macromolecules in these venous wall compartments during the adaptive response of vein grafts to the arterial circulation.