Tissue-engineered Vascular Constructs Research Articles

Vascular smooth muscle cells derived from inbred swine induced pluripotent stem cells for vascular tissue engineering.

Development of autologous tissue-engineered vascular constructs using vascular smooth muscle cells (VSMCs) derived from human induced pluripotent stem cells (iPSCs) holds great potential in treating patients with vascular disease. However, preclinical, large animal iPSC-based cellular and tissue models are required to evaluate safety and efficacy prior to clinical application. Herein, swine iPSC (siPSC) lines were established by introducing doxycycline-inducible reprogramming factors into fetal fibroblasts from a line of inbred Massachusetts General Hospital miniature swine that accept tissue and organ transplants without immunosuppression within the line. Highly enriched, functional VSMCs were derived from siPSCs based on addition of ascorbic acid and inactivation of reprogramming factor via doxycycline withdrawal. Moreover, siPSC-VSMCs seeded onto biodegradable polyglycolic acid (PGA) scaffolds readily formed vascular tissues, which were implanted subcutaneously into immunodeficient mice and showed further maturation revealed by expression of the mature VSMC marker, smooth muscle myosin heavy chain. Finally, using a robust cellular self-assembly approach, we developed 3D scaffold-free tissue rings from siPSC-VSMCs that showed comparable mechanical properties and contractile function to those developed from swine primary VSMCs. These engineered vascular constructs, prepared from doxycycline-inducible inbred siPSCs, offer new opportunities for preclinical investigation of autologous human iPSC-based vascular tissues for patient treatment.

Design and evaluation of a novel subatmospheric pressure bioreactor for the preconditioning of tissue-engineered vascular constructs.

The pre-conditioning of tissue-engineered vascular scaffolds with mechanical stimuli is being recognised as an essential step in producing a functional vascular construct. In this study we design and evaluate a novel bioreactor, which exerts a mechanical strain on developing vascular scaffolds via subatmospheric pressure. We design and construct a bioreactor, which exerts subatmospheric pressure via a vacuum assisted closure unit. Vascular scaffolds seeded with human umbilical endothelial cells were evaluated for structural integrity, microbial contamination, cellular viability, von Willebrand factor (VWF) production, cell proliferation and morphology under a range of subatmospheric pressures (75-200mmHg). The bioreactor produced sustained subatmospheric pressures, which exerted a mechanical strain on the vascular scaffold. No microbial contamination was found during the study. The structural integrity of the vascular construct was maintained. There was no difference in cellular viability between control or subatmospheric pressure groups (p = 0.817). Cells continued to produce VWF under a range of subatmospheric pressures. Cells subjected to subatmospheric pressures of 125mmHg and 200mmHg exhibited higher levels of growth than cells in atmospheric pressure at 24 (p≤0.016) and 48 hour (p≤0.001). Negative pressure affected cellular morphology, which were more organised, elongated and expanded when exposed to subatmospheric pressure. We have constructed and validated a novel subatmospheric bioreactor. The bioreactor maintained a continuous subatmospheric pressure to the vascular scaffolds in a stable, sterile and constant environment. The bioreactor exerted a strain on the vascular sheets, which was shown to alter cellular morphology and enhance cellular proliferation.

A Dynamically Cultured Collagen/Cells-Incorporated Elastic Scaffold for Small-Diameter Vascular Grafts

There is an essential demand for tissue-engineered autologous small-diameter vascular grafts, which offer temporary supports and guides for vascular tissue organization, repair and remodeling. This study reports on the effect of collagen/smooth muscle cells (SMCs) mixtures under dynamic cultures and SMC-endothelial cell (ECs) co-culture on cell proliferation, uniform cell distribution, extracellular matrix deposition, and endothelial cells monolayer formation in tissue-engineered tubular arterial constructs of 4 mm inner diameter. Rabbit aortic SMCs were infiltrated with collagen solution in poly(L-lactide-co-ϵ-caprolactone) (PLCL) scaffolds under vacuum to form collagenous gel and subjected to dynamic strain by culturing them in a dynamic perfusion bioreactor. The construct lumen was subsequently seeded with ECs and experiments were completed to create ECs–SMCs co-culture constructs. The collagen/SMCs incorporated elastic scaffold cultured under dynamic culture conditions promoted matrix deposition, leading to the development of tissue-engineered vascular constructs, and induced SMC to have more uniform cell distribution. Scanning electron microscopic examination and von Willebrand Factor staining demonstrated the presence of ECs spread over the lumen. Quantitative analysis of elastin contents demonstrated that the engineered vessels acquired similar elastin contents as native arteries. The collagen/SMCs/ECs incorporated PLCL scaffolds under dynamic culture conditions can be used as a scaffold for tissue engineering to facilitate small-diameter vascular-tissue formation.

Mechanical Properties of Tissue-Engineered Vascular Constructs Produced Using Arterial or Venous Cells

There is a clinical need for better blood vessel substitutes, as current surgical procedures are limited by the availability of suitable autologous vessels and suboptimal behavior of synthetic grafts in small caliber arterial graft (<5 mm) applications. The aim of the present study was to compare the mechanical properties of arterial and venous tissue-engineered vascular constructs produced by the self-assembly approach using cells extracted from either the artery or vein harvested from the same human umbilical cord. The production of a vascular construct comprised of a media and an adventitia (TEVMA) was achieved by rolling a continuous tissue sheet containing both smooth muscle cells and adventitial fibroblasts grown contiguously in the same tissue culture plate. Histology and immunofluorescence staining were used to evaluate the structure and composition of the extracellular matrix of the vascular constructs. The mechanical strength was assessed by uniaxial tensile testing, whereas viscoelastic behavior was evaluated by stepwise stress-relaxation and by cyclic loading hysteresis analysis. Tensile testing showed that the use of arterial cells resulted in stronger and stiffer constructs when compared with those produced using venous cells. Moreover, cyclic loading demonstrated that constructs produced using arterial cells were able to bear higher loads for the same amount of strain when compared with venous constructs. These results indicate that cells isolated from umbilical cord can be used to produce vascular constructs. Arterial constructs possessed superior mechanical properties when compared with venous constructs produced using cells isolated from the same human donor. This study highlights the fact that smooth muscle cells and fibroblasts originating from different cell sources can potentially lead to distinct tissue properties when used in tissue engineering applications.

A Computer-Controlled Apparatus for the Characterization of Mechanical and Viscoelastic Properties of Tissue-Engineered Vascular Constructs

Tissue-engineered blood vessels can be partly characterized by analyzing their mechanical properties using burst pressure testing, compliance measurement, creep and cyclic testing. Studying these parameters provides information on the capability of a fabrication method to produce tissue-engineered blood vessels (TEBV) and allow for the optimization of their resistance and viscoelastic properties. This study presents the design and fabrication of an apparatus allowing accurate and reliable measurements of the mechanical properties of tissue-engineered vascular constructs. A computer-controlled system was designed to monitor pressure and diameter variations of vascular constructs submitted to hydrostatic loading. The system was programmed to control the motorized portion of the setup and allow simultaneous data acquisition, analysis and real-time display. Data acquisition cards allow for synchronous monitoring of pressure and diameter of the constructs through a pressure transducer and a CCD camera. Image analysis and pressure data computation resulted in compliance, creep and dynamic characterization of the tested tissues. This experimental setup succeeded in measuring the burst pressure, compliance, creep and cyclic behavior of tissue-engineered vascular media (TEVM), adventitia (TEVA) and a combination of a media and an adventitia (TEVMA) reconstructed by the self-assembly method. Our apparatus has proven to be a precise and reliable tool for the characterization of the mechanical properties of vascular constructs.

A Novel Single-Step Self-Assembly Approach for the Fabrication of Tissue-Engineered Vascular Constructs

There is a clinical need for a functional tissue-engineered blood vessel because small-caliber arterial graft (<5 mm) applications are limited by the availability of suitable autologous vessels and suboptimal performances of synthetic grafts. This study presents an analysis of the mechanical properties of tissue-engineered vascular constructs produced using a novel single-step self-assembly approach. Briefly, the tissue-engineered vascular media were produced by culturing smooth muscle cell in the presence of sodium l-ascorbate until the formation of a cohesive tissue sheet. This sheet was then rolled around a tubular support to create a media construct. Alternatively, the tissue-engineered vascular adventitia was produced by rolling a tissue sheet obtained from dermal fibroblasts or saphenous vein fibroblasts. The standard self-assembly approach to obtain the two-layer tissue-engineered vascular constructs comprising both media and adventitia constructs consists of two steps in which tissue-engineered vascular media were first rolled on a tubular support and a tissue-engineered vascular adventitia was then rolled on top of the first layer. This study reports an original alternative method for assembling tissue-engineered vascular constructs comprising both media and an adventitia in a single step by rolling a continuous tissue sheet containing both cell types contiguously. This tissue sheet was produced by growing smooth muscle cells alongside fibroblasts (saphenous vein fibroblasts or dermal fibroblasts) in the same culture dish separated by a spacer, which is removed later in the culture period. The mechanical strength assessed by uniaxial tensile testing, burst pressure measurements, and viscoelastic behavior evaluated by stepwise stress relaxation tests reveals that the new single-step fabrication method significantly improves the mechanical properties of tissue-engineered vascular construct for both ultimate tensile strength and all the viscoelastic moduli.

Applications of Human Tissue-Engineered Blood Vessel Models to Study the Effects of Shed Membrane Microparticles from T-Lymphocytes on Vascular Function

Microparticles (MPs) are membrane vesicles harboring cell surface proteins and containing cytoplasmic components of the original cell. High levels of circulating MPs have been detected in pathological states associated with vascular dysfunction. We took advantage of the self-assembly method of tissue engineering to produce in vitro three vascular constructs from human vascular smooth muscle cells and fibroblasts to investigate the role of the adventitia in the modulation of vascular tone by MPs, comparing the contractile response of each of these constructs to histamine. The first two were composed of an adventitia (tissue-engineered vascular adventitia (TEVA)) or a media (tissue-engineered vascular media (TEVM)) solely, and the third one contained a media and an adventitia (tissue-engineered vascular media and adventitia (TEVMA)). In the three constructs, the results show that histamine induces contraction insensitive to blockade of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2) and not affected by MP treatment. MPs decreased NO production and nuclear factor (NF)-kappaB expression but did not affect superoxide anion (O(2)(-)) release in TEVA. MPs enhanced NF-kappaB expression but did not affect iNOS and COX-2 expression or NO or O(2)(-) release in TEVM. In TEVMA, MPs did not enhance NF-kappaB expression, but COX-2 expression was higher, and O(2)(-) release was lower. Thus, MPs affected NO, O(2)(-), NF-kappaB, and COX-2 in a subtle fashion to maintain the contractile response to histamine. The use of tissue-engineered vascular constructs results in a better understanding of the effect of MPs on human adventitia and media.

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo. Microfluidic micropatterning was used to create 15-microm wide adhesive lanes with 45-microm spacing to reproduce the elongated EC morphology without the influence of external forces. Both EPCs and ECs elongated on micropatterned lanes had aligned actin cytoskeleton and readily deposited ECM. EPCs deposited and remodeled the ECM to a greater extent than ECs. Since a readily produced ECM can improve graft patency, EPCs are an advantageous cell source for endothelializing vascular constructs. Furthermore, EC deposition of ECM was dependent on cell morphology, where elongated ECs deposited more collagen IV and less fibronectin compared to matched cobblestone controls. Thus micropatterned surfaces controlled EC shape and ECM deposition, which ultimately has implications for the design of tissue-engineered vascular constructs.

A Novel Pulsatile, Laminar Flow Bioreactor for the Development of Tissue-Engineered Vascular Structures

Exposure of vascular cell-seeded, tubular, biodegradable polymers to pulsatile flow conditions has been proposed as a method to develop tissue-engineered blood vessels by "maturing" structural integrity, and increasing collagen content, suture retention, burst pressure, and tissue formation. These in vitro tissue-engineered arteries demonstrate contractile responses to pharmacologic agents and express markers of vascular differentiation. Current methods to induce pulsatile flow in a bioreactor system are limited by the creation of nonphysiologic pressure waveforms and noncompliant reservoirs to house the tissue-engineered vascular constructs. We have developed a novel method for the in vitro development of tubular vascular structures by using a mechanical ventilator to induce pulsatile, laminar flow into a fluid column, resulting in pressurized waveforms similar to mammalian physiology. The vascular constructs are housed in semicompliant tubing to facilitate an additional variable of circumferential stretch as a potential signaling mechanism. This approach more closely approximates mammalian physiology and we hypothesize that it will facilitate mechanical signaling necessary for the development of tissue-engineered vessels for clinical applications.