Harnessing topographical & biochemical cues to enhance elastogenesis by paediatric cells for cardiovascular tissue engineering applications.

The development of tissue-engineered vascular grafts (TEVGs) with a biomimetic extracellular matrix (ECM) structure, including a mature elastic network, remains a key challenge for the production of grafts with long-term functionality. The aim of this study was to investigate the influence of aligned nanofiber substrates on ECM protein synthesis by neonatal smooth muscle cells (SMCs), and to examine the combined effects of this topographical cue in conjunction with transforming growth factor beta-1 (TGF-ß1) - a biochemical elastogenic promoter. Glass coverslips were coated in electrospun fibrinogen nanofibers (average diameter < 500 nm) with either a randomly-orientated or aligned topography. Human umbilical artery smooth muscle cells (hUASMCs) were cultured on the electrospun substrates for 7 and 14 days, with or without a 2 ng/ml TGF-ß1 supplement. The ECM structure was analysed using immunohistochemistry and the quantity of secreted elastin in the cell layer was measured using a dye-binding assay. Aligned fiber substrates induced a directed orientation of both the seeded cells and cell-synthesized ECM fibers. Cells cultured on aligned fibers exhibited a significant increase in the expression of phenotypic contractile proteins, as well as increases in the secreted elastin content of the cell layer, compared to cells cultured on randomly-orientated substrates. TGF-ß1 supplementation was shown to synergistically increase secreted elastin from cells cultured on aligned fiber substrates (p < 0.05). Aligned nanofiber scaffolds can be used to direct cellular orientation, elastin-related protein synthesis and cell phenotype, and consequently there is potential for their application in the development of TEVGs as part of a multi-pronged strategy to promote elastic fiber formation.

Surface-sensitive two-dimensional magneto-fingerprint in mesoscopic Bi2Se3 channels.

Periodic Aharonov­Bohm and Altshuler­Aronov­Spivak oscillations have traditionally been observed in lateral transport through patterned mesoscopic loops of diffusive conductors. However, our studies of perpendicular-to-plane magnetotransport in straight-channel, diffusive devices of epitaxial Bi2Se3 surprisingly reveal signatures of Aharonov­Bohm orbits, periodic conductance fluctuation magneto-fingerprints, even though the devices are not explicitly patterned into loops. We show that the length scale of these orbits corresponds to the typical perimeter of triangular terraces found on the surface of these thin film devices, strongly suggesting that the periodic magneto-fingerprint arises from coherent scattering of electron waves from the step-edges. Our interpretation is bolstered by control measurements in devices without such surface morphology that only show a conventional, aperiodic magneto-fingerprint. These results show that lithographically patterned Bi2Se3 devices provide a novel class of mesoscopic physical systems for systematic studies of coherent surface sensitive transport.

Fabrication of blood-derived elastogenic vascular grafts using electrospun fibrinogen and polycaprolactone composite scaffolds for paediatric applications.

The development of tissue-engineered vascular grafts (TEVGs) for paediatric applications must consider unique factors associated with this patient cohort. Although the increased elastogenic potential of neonatal cells offers an opportunity to overcome the long-standing challenge of in vitro elastogenesis, neonatal patients have a lower tolerance for autologous tissue harvest and require grafts that exhibit growth potential. The purpose of this study was to apply a multipronged strategy to promote elastogenesis in conjunction with umbilical cord-derived materials in the production of a functional paediatric TEVG. An initial proof-of-concept study was performed to extract fibrinogen from human umbilical cord blood samples and, through electrospinning, to produce a nanofibrous fibrinogen scaffold. This scaffold was seeded with human umbilical artery-derived smooth muscle cells (hUASMCs), and neotissue formation within the scaffold was examined using immunofluorescence microscopy. Subsequently, a polycaprolactone-reinforced porcine blood-derived fibrinogen scaffold (isolated using the same protocol as cord blood fibrinogen) was used to develop a rolled-sheet graft that employed topographical and biochemical guidance cues to promote elastogenesis and cellular orientation. This approach resulted in a TEVG with robust mechanical properties and biomimetic arrangement of extracellular matrix (ECM) with rich expression of elastic fibre-related proteins. The results of this study hold promise for further development of paediatric TEVGs and the exploration of the effects of scaffold microstructure and nanostructure on vascular cell function and ECM production.

A biomimetic urethral model to evaluate urinary catheter lubricity and epithelial micro-trauma.

The standard method of evaluating the lubricity of intermittent urinary catheters with coefficient of friction (CoF) testing is not physiologically relevant, while there is also a dearth of published research on catheter-associated urethral micro-trauma. We developed a novel human urethral epithelial cell-seeded model of the urethra to replace the rubber counter-surface used in standard CoF testing. This cell-seeded model, in conjunction with a novel testing device, allows an investigation of catheter-associated epithelial micro-trauma in vitro for the first time. The CoF of four brands of commercially-available hydrophilic-coated intermittent catheters was measured using both the rubber and urethral model counter-surfaces. Post-catheterisation of the urethral model, the damage to the epithelial layer was analysed using standard cell imaging. The rubber counter-surface was shown to over-estimate the CoF of gel-coated catheters compared to our urethral model due to stick-slip behaviour caused by polymer-on-polymer interaction of the catheter base material on the rubber counter-surface. We identified no deleterious effect due to the presence or design of catheter eyelets to either the CoF measurements or the degree of epithelium damage in our model. Furthermore, the epithelial damage did not correlate with the measured CoF of the low friction catheters, suggesting a more nuanced pathogenesis of urethral irritation and casting doubt on the translatability of a solely mechanical assessment of lubricity of urinary catheters to a clinical effect.

Development of a composite degradable/nondegradable tissue-engineered vascular graft.

The present study aimed to determine the feasibility of constructing a reinforced autologous vascular graft by combining the advantages of fibrin gel as an autologous cell carrier material with the inherent mechanical strength of an integrated mesh structure. It was hypothesized that the mesh and dynamic culture conditions could be combined to generate mechanically stable and implantable vascular grafts within a shorter cultivation period than traditional methods. A two-step moulding technique was developed to integrate a polyvinylidene fluoride (PVDF) mesh (pore size: 1-2 mm) in the wall of a fibrin-based vascular graft (I.D. 5 mm) seeded with carotid myofibroblasts. The graft was cultured under increasing physiological flow conditions for 2 weeks. Histology, burst strength, and suture retention strength were evaluated. Cell growth and tissue development was excellent within the fibrin gel matrix surrounding the PVDF fibers, and tissue structure demonstrated remarkable similarity to native tissue. The grafts were successfully subjected to physiological flow rates and pressure gradients from the outset, and mechanical properties were enhanced by the mesh structure. Mean suture retention strength of the graft tissue was 6.3 N and the burst strength was 236 mm Hg. Using the vascular composite graft technique, the production of tissue engineered, small-caliber vascular grafts with good mechanical properties within a conditioning period of 14 days is feasible.

The in vitro development of autologous fibrin-based tissue-engineered heart valves through optimised dynamic conditioning.

Our group has previously demonstrated the synthesis of a completely autologous fibrin-based heart valve structure using the principles of tissue engineering. The present approach aims to guide more mature tissue development in fibrin-based valves based on in vitro conditioning in a custom-designed bioreactor system. Moulded fibrin-based tissue-engineered heart valves seeded with ovine carotid artery-derived cells were subjected to 12 days of mechanical conditioning in a bioreactor system. The bioreactor pulse rate was increased from 5 to 10 b.p.m. after 6 days, while a pressure difference of 20 mmH(2)O was maintained over the valve leaflets. Control valves were cultured under stirred conditions in a beaker. Cell phenotype and extracellular matrix (ECM) composition were analysed in all samples and compared to native ovine aortic valve tissue using routine histological and immunohistochemical techniques. Conditioned valve leaflets showed reduced tissue shrinkage compared to stirred controls. Limited ECM synthesis was evident in stirred controls, while the majority of cells were detached from the fibrin scaffold. Dynamic conditioning increased cell attachment/alignment and expression of alpha-smooth muscle actin, while enhancing the deposition of ECM proteins, including types I and III collagen, fibronectin, laminin and chondroitin sulphate. There was no evidence for elastin synthesis in either stirred controls or conditioned samples. The present study demonstrates that the application of low-pressure conditions and increasing pulsatile flow not only enhances seeded cell attachment and alignment within fibrin-based heart valves, but dramatically changes the manner in which these cells generate ECM proteins and remodel the valve matrix. Optimised dynamic conditioning, therefore, might accelerate the maturation of surgically feasible and implantable autologous fibrin-based tissue-engineered heart valves.

Biomechanical analyses of overlap and mesh dislocation in an incisional hernia model in vitro.

BACKGROUND: Incisional hernia repair is one of the most common surgical complications. Despite the introduction of mesh techniques of repair, recurrences are still prevalent. The aim of the current study was to evaluate the dependence of mesh dislocation on defect size, facial overlap, mesh-position, and orientation of the mesh in cases of anisotropic stretchability. METHODS: An in vitro incisional hernia model was used, which consisted of a pressure chamber, an elastic silicone pad representing the peritoneal sac, and a silicone mat with bovine muscle tissue representing the abdominal wall. Intrinsic pressure (up to 200 mm Hg) was generated within the pressure chamber by continuous inflation with CO(2). A slit-like or flap-like defect was created in the silicone mat to simulate small or large hernia defects, respectively. The implanted mesh was arranged in both onlay and sublay configurations. A large pore polypropylene mesh with significant anisotropic stretchability was investigated, whereas overlaps of 2, 3, and 4 cm were applied. RESULTS: Despite the application of pressures up to 200 mm Hg, no mesh ruptures occurred. In the slit-like defect model, the minimal overlap required to prevent dislocation at 200 mm Hg was 3 cm using the sublay technique provided that the mesh was positioned with its most stretchable axis parallel to the largest slit dehiscence. Perpendicular rotation of the mesh resulted in dislocation at 160 mm Hg, despite using an overlap of 3 cm. Mesh reinforcement showed less stability in both the onlay position and the flap-like defect. CONCLUSION: An overlap of 3 cm is sufficient to prevent early mesh dislocation. Meshes with anisotropic stretchability should be orientated with the most stretchable axis in the direction of least overlap.

Helicity dependent photocurrent in electrically gated (Bi1-x Sb x )2Te3 thin films.

Circularly polarized photons are known to generate a directional helicity-dependent photocurrent in three-dimensional topological insulators at room temperature. Surprisingly, the phenomenon is readily observed at photon energies that excite electrons to states far above the spin-momentum locked Dirac cone and the underlying mechanism for the helicity-dependent photocurrent is still not understood. Here we show a comprehensive study of the helicity-dependent photocurrent in (Bi1-x Sb x )2Te3 thin films as a function of the incidence angle of the optical excitation, its wavelength and the gate-tuned chemical potential. Our observations allow us to unambiguously identify the circular photo-galvanic effect as the dominant mechanism for the helicity-dependent photocurrent. Additionally, we use an analytical model to relate the directional nature of the photocurrent to asymmetric optical transitions between the topological surface states and bulk bands. The insights we obtain are important for engineering opto-spintronic devices that rely on optical steering of spin and charge currents.

Freeze-Drying as a Novel Biofabrication Method for Achieving a Controlled Microarchitecture within Large, Complex Natural Biomaterial Scaffolds.

The biofabrication of large natural biomaterial scaffolds into complex 3D shapes which have a controlled microarchitecture remains a major challenge. Freeze-drying (or lyophilization) is a technique used to generate scaffolds in planar 3D geometries. Here we report the development of a new biofabrication process to form a collagen-based scaffold into a large, complex geometry which has a large height to width ratio, and a controlled porous microarchitecture. This biofabrication process is validated through the successful development of a heart valve shaped scaffold, fabricated from a collagen-glycosaminoglycan co-polymer. Notably, despite the significant challenges in using freeze-drying to create such a structure, the resultant scaffold has a uniform, homogenous pore architecture throughout. This is achieved through optimization of the freeze-drying mold and the freezing parameters. We believe this to be the first demonstration of using freeze-drying to create a large, complex scaffold geometry with a controlled, porous architecture for natural biomaterials. This study validates the potential of using freeze-drying for development of organ-specific scaffold geometries for tissue engineering applications, which up until now might not have been considered feasible.

A collagen-glycosaminoglycan co-culture model for heart valve tissue engineering applications.

In order to develop efficient design strategies for a tissue-engineered heart valve, in vivo and in vitro models of valvular structure and cellular function require extensive characterisation. Collagen and glycosaminoglycans (GAGs) provide unique functional characteristics to the heart valve structure. In the current study, type I collagen-GAG hydrogels were investigated as biomaterials for the creation of mitral valve tissue. Porcine mitral valve interstitial cells (VICs) and endothelial cells (VECs) were isolated and co-cultured for 4 weeks in hydrogel constructs composed of type I collagen. The metabolic activity and tissue organisation of mitral valve tissue constructs was evaluated in the presence and absence of chondroitin sulphate (CS) GAG, and comparisons were made with normal mitral valve tissue. Both collagen and collagen-CS mitral valve constructs contracted to form tissue-like structures in vitro. Biochemical assay demonstrated that over 75% of CS was retained within collagen-CS constructs. Morphological examination demonstrated enhanced VEC surface coverage in collagen-CS constructs compared to collagen constructs. Ultrastructural analysis revealed basement membrane synthesis and cell junction formation by construct VECs, with an increased matrix porosity observed in collagen-CS constructs. Immunohistochemical analyses demonstrated enhanced extracellular matrix production in collagen-CS constructs, including expression of elastin and laminin by VICs. Both native valve and collagen-CS construct VECs also expressed the vasoactive molecule, eNOS, which was absent from collagen construct VECs. The present study demonstrates that collagen gels can be used as matrices for the in vitro synthesis of tissue structures resembling mitral valve tissue. Addition of CS resulting in a more porous model was shown to positively influence the bioactivity of seeded valve cells and tissue remodelling. Collagen-GAG matrices may hold promise for a potential use in heart valve tissue engineering and improved understanding of heart valve biology.

Incorporation of fibrin into a collagen-glycosaminoglycan matrix results in a scaffold with improved mechanical properties and enhanced capacity to resist cell-mediated contraction.

Fibrin has many uses as a tissue engineering scaffold, however many in vivo studies have shown a reduction in function resulting from the susceptibility of fibrin to cell-mediated contraction. The overall aim of the present study was to develop and characterise a reinforced natural scaffold using fibrin, collagen and glycosaminoglycan (FCG), and to examine the cell-mediated contraction of this scaffold in comparison to fibrin gels. Through the use of an injection loading technique, a homogenous FCG scaffold was developed. Mechanical testing showed a sixfold increase in compressive modulus and a thirtyfold increase in tensile modulus of fibrin when reinforced with a collagen-glycosaminoglycan backbone structure. Human vascular smooth muscle cells (vSMCs) were successfully incorporated into the FCG scaffold and demonstrated excellent viability over 7 days, while proliferation of these cells also increased significantly. VSMCs were seeded into both FCG and fibrin-only gels at the same seeding density for 7 days and while FCG scaffolds did not demonstrate a reduction in size, fibrin-only gels contracted to 10% of their original diameter. The FCG scaffold, which is composed of natural biomaterials, shows potential for use in applications where dimensional stability is crucial to the functionality of the tissue. STATEMENT OF SIGNIFICANCE: Fibrin is a versatile scaffold for tissue engineering applications, but its weak mechanical properties leave it susceptible to cell-mediated contraction, meaning the dimensions of the fibrin construct will change over time. We have reinforced fibrin with a collagen glycosaminoglycan matrix and characterised the mechanical properties and bioactivity of the reinforced fibrin (FCG). This is the first scaffold manufactured from all naturally derived materials that resists cell-mediated contraction. In fact, over 7 days, the FCG scaffold fully resisted cell-mediated contraction of vascular smooth muscle cells. This FCG scaffold has many potential applications where natural scaffold materials can encourage regeneration.

Electrospinning of biomimetic scaffolds for tissue-engineered vascular grafts: threading the path.

Tissue-engineered vascular grafts (TEVGs) offer an alternative to synthetic grafts for the surgical treatment of atherosclerosis and congenital heart defects, and may improve graft patency and patient outcomes after implantation. Electrospinning is a versatile manufacturing process for the production of fibrous scaffolds. This review aims to investigate novel approaches undertaken to improve the design of electrospun scaffolds for TEVG development. The review describes how electrospinning can be adapted to produce aligned nanofibrous scaffolds used in vascular tissue engineering, while novel processes for improved performance of such scaffolds are examined and compared to evaluate their effectiveness and potential. By highlighting new drug delivery techniques and porogenic technologies, in addition to analyzing in vitro and in vivo testing of electrospun TEVGs, it is hoped that this review will provide guidance on how the next generation of electrospun vascular graft scaffolds will be designed and tested for the potential improvement of cardiovascular therapies.

Ovine carotid artery-derived cells as an optimized supportive cell layer in 2-D capillary network assays.

BACKGROUND: Endothelial cell co-culture assays are differentiation assays which simulate the formation of capillary-like tubules with the aid of a supportive cell layer. Different cell types have been employed as a supportive cell layer, including human pulmonary artery smooth muscle cells (PASMCs) and human mammary fibroblasts. However, these sources of human tissue-derived cells are limited, and more readily accessible human or animal tissue-derived cell sources would simplify the endothelial cell co-culture assay. In the present study, we investigated the potential use of alternative, accessible supportive cells for endothelial cell co-culture assay, including human umbilical cord and ovine carotid artery. METHODS AND RESULTS: Human umbilical artery SMCs (HUASMCs) and ovine carotid artery-derived cells were seeded into 96-well plates, followed by addition of human umbilical vein endothelial cells (HUVECs). Nine days after co-culture, cells were fixed, immunostained and analysed using an in vitro angiogenesis quantification tool. Capillary-like structures were detected on ovine carotid artery-derived supportive cell layers. The initial cell number, as well as pro- and anti-angiogenic factors (VEGF, PDGF-BB and Bevacizumab), had a positive or negative influence on the number of capillary-like structures. Furthermore, HUVECs from different donors showed distinct levels of VEGF receptor-2, which correlated with the amount of capillary-like structures. In the case of HUASMC supportive cell layers, HUVECs detached almost completely from the surface. CONCLUSIONS: Cells of different origin have a varying applicability regarding the endothelial cell co-culture assay: under the conditions described here, ovine carotid artery-derived cells seem to be more suitable than HUASMCs for an endothelial co-culture assay. Furthermore, the ovine carotid artery-derived cells are easier to obtain and are in more abundant supply than the currently used dermal or breast tissue cells. The use of ovine carotid artery-derived cells simplifies the endothelial co-culture assay with respect to testing large amounts of pro- and anti-angiogenic factors.

Biofunctionalized microfiber-assisted formation of intrinsic three-dimensional capillary-like structures.

OBJECTIVES: A vascular supply network is essential in engineered tissues >100-200-µm thickness. To control vascular network formation in vitro, we hypothesize that capillarization can be achieved locally by using fibers to position and guide vessel-forming endothelial cells within a three-dimensional (3D) matrix. MATERIALS AND METHODS: Biofunctionalization of poly-(L-lactic acid) (PLLA) fibers was performed by amino-functionalization and covalent binding of RGD peptides. Human foreskin fibroblasts (HFFs) and human umbilical vein endothelial cells (HUVECs) were seeded on the fibers in a mould and subsequently embedded in fibrin gel. After 9-21 days of coculture, constructs were fixed and immunostained (PECAM-1). Capillary-like structures with lumen in the 3D fibrin matrix were verified and quantified using two-photon microscopy and image analysis software. RESULTS: Capillary-like networks with lumen formed adjacent to the PLLA fibers. Increased cell numbers were observed to attach to RGD-functionalized fibers, resulting in enhanced formation of capillary-like structures. Cocultivation of HFFs sufficiently supported HUVECs in the formation of capillary-like structures, which persisted for at least 21 days of coculture. CONCLUSIONS: The guidance of vessel growth within tissue-engineered constructs can be achieved using biofunctionalized PLLA microfibers. Further methods are warranted to perform specified spatial positioning of fibers within 3D formative scaffolds to enhance the applicability of the concept.

Influence of platelet-derived growth factor-AB on tissue development in autologous platelet-rich plasma gels.

Fibrin-based scaffolds are widely used in tissue engineering. We postulated that the use of platelet-rich plasma (PRP) in contrast to platelet-poor plasma and pure fibrinogen as the basic material leads to an increased release of autologous platelet-derived growth factor (PDGF)-AB, which may have a consequent positive effect on tissue development. Therefore, we evaluated the release of PDGF-AB during the production process and the course of PDGF release during cultivation of plasma gels with and w/o platelets. The influence of PDGF-AB on the proliferation rate of human umbilical cord artery smooth muscle cells (HUASMCs) was studied using XTT assay. The synthesis of extracellular matrix by HUASMCs in plasma- and fibrin gels was measured using hydroxyproline assay. The use of PRP led to an increase in autologous PDGF-AB release. Further, the platelet-containing plasma gels showed a prolonged release of growth factor during cultivation. Both PRP and platelet-poor plasma gels had a positive effect on the production of collagen. However, PDGF-AB as a supplement in medium and in pure fibrin gel had neither an effect on cell proliferation nor on the collagen synthesis rate. This observation may be due to an absence of PDGF receptors in HUASMCs as determined by flow cytometry. In conclusion, although the prolonged autologous production of PDGF-AB in PRP gels is possible, the enhanced tissue development by HUASMCs within such gels is not PDGF related.

Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation.

There is a clear clinical requirement for the design and development of living, functional, small-calibre arterial grafts. Here, we investigate the potential use of a small diameter, tissue-engineered artery in a pre-clinical study in the carotid artery position of sheep. Small-calibre ( approximately 5 mm) vascular composite grafts were molded using a fibrin scaffold supported by a poly(L/D)lactide 96/4 (P(L/D)LA 96/4) mesh, and seeded with autologous arterial-derived cells prior to 28 days of dynamic conditioning. Conditioned grafts were subsequently implanted for up to 6 months as interposed carotid artery grafts in the same animals from which the cells were harvested. Explanted grafts (n = 6) were patent in each of the study groups (1 month, 3 months, 6 months), with a significant stenosis in one explant (3 months). There was a complete absence of thrombus formation on the luminal surface of grafts, with no evidence for aneurysm formation or calcification after 6 months in vivo. Histological analyses revealed remodeling of the fibrin scaffold with mature autologous proteins, and excellent cell distribution within the graft wall. Positive vWf and eNOS staining, in addition to scanning electron microscopy, revealed a confluent monolayer of endothelial cells lining the luminal surface of the grafts. The present study demonstrates the successful production and mid-term application of an autologous, fibrin-based small-calibre vascular graft in the arterial circulation, and highlights the potential for the creation of autologous implantable arterial grafts in a number of settings.

Tranexamic acid--an alternative to aprotinin in fibrin-based cardiovascular tissue engineering.

Recent clinical trials have led to the worldwide suspension of aprotinin, the most commonly used antifibrinolytic agent in fibrin-based tissue engineering. For future clinical applications of fibrin-based scaffolds, a suitable, alternative fibrinolysis inhibitor must be identified. The present study aimed to evaluate tranexamic acid (trans-4-aminomethyl-cyclohexane-1-carboxylic acid [t-AMCA]) as an alternative fibrinolysis inhibitor to aprotinin for cardiovascular tissue engineering applications. The effects of various concentrations of t-AMCA (30-160 microg/mL) and aprotinin on fibrin gel-lysis were spectrophotometrically quantified in vitro. Cytotoxic effects of t-AMCA and aprotinin on carotid artery-derived cells, in addition to their influence on fibrin gel mechanical strength, were examined. Further, the influence of t-AMCA versus aprotinin on three-dimensional fibrin-based constructs was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. The results demonstrated that neither t-AMCA (30-160 microg/mL) nor aprotinin elicited cytotoxic effects on cultured cells. Although aprotinin showed reduced fibrinolysis in the presence of plasmin compared to t-AMCA, no significant difference was obtained under standard culture conditions. Additionally, t-AMCA had no negative influence on the mechanical stability of fibrin gels, which also demonstrated excellent cell morphology, tissue development, and ultrastructure. The results from the present study demonstrate that t-AMCA may be a suitable alternative to aprotinin for controlling the in vitro degradation rate of fibrin-based tissue-engineered constructs.

In vivo remodeling and structural characterization of fibrin-based tissue-engineered heart valves in the adult sheep model.

Autologous fibrin-based tissue-engineered heart valves have demonstrated excellent potential as patient-derived valve replacements. The present pilot study aims to evaluate the structure and mechanical durability of fibrin-based heart valves after implantation in a large-animal model (sheep). Tissue-engineered heart valves were molded using a fibrin scaffold and autologous arterial-derived cells before 28 days of mechanical conditioning. Conditioned valves were subsequently implanted in the pulmonary trunk of the same animals from which the cells were harvested. After 3 months in vivo, explanted valve conduits (n = 4) had remained intact and exhibited native tissue consistency, although leaflets demonstrated insufficiency because of tissue contraction. Routine histology showed remarkable tissue development and cell distribution, along with functional blood vessel ingrowth. A confluent monolayer of endothelial cells was present on the valve surface, as evidenced by scanning electron microscopy and positive von Willebrand factor staining. Immunohistochemistry and extracellular matrix (ECM) assay demonstrated complete resorption of the fibrin scaffold and replacement with ECM proteins. Transmission electron microscopy revealed mature collagen formation and viable, active resident tissue cells. The preliminary findings of implanted fibrin-based tissue-engineered heart valves are encouraging, with excellent tissue remodeling and structural durability after 3 months in vivo. The results from this pilot study highlight the potential for construction of completely "autologous" customized tissue-engineered heart valves based on a patient-derived fibrin scaffold.

Tissue-engineered small-caliber vascular graft based on a novel biodegradable composite fibrin-polylactide scaffold.

Small-caliber vascular grafts (< or =5 mm) constructed from synthetic materials for coronary bypass or peripheral vascular repair below the knee have poor patency rates, while autologous vessels may not be available for harvesting. The present study aimed to create a completely autologous small-caliber vascular graft by utilizing a bioabsorbable, macroporous poly(L/D)lactide 96/4 [P(L/D)LA 96/4] mesh as a support scaffold system combined with an autologous fibrin cell carrier material. A novel molding device was used to integrate a P(L/D)LA 96/4 mesh in the wall of a fibrin-based vascular graft, which was seeded with arterial smooth muscle cells (SMCs)/fibroblasts and subsequently lined with endothelial cells. The mold was connected to a bioreactor circuit for dynamic mechanical conditioning of the graft over a 21-day period. Graft cell phenotype, proliferation, extracellular matrix (ECM) content, and mechanical strength were analyzed. alpha-SMA-positive SMCs and fibroblasts deposited ECM proteins into the graft wall, with a significant increase in both cell number and collagen content over 21 days. A luminal endothelial cell lining was evidenced by vWf staining, while the grafts exhibited supraphysiological burst pressure (>460 mmHg) after dynamic cultivation. The results of our study demonstrated the successful production of an autologous, biodegradable small-caliber vascular graft in vitro, with remodeling capabilities and supraphysiological mechanical properties after 21 days in culture. The approach may be suitable for a variety of clinical applications, including coronary artery and peripheral artery bypass procedures.