Successful Development of Small Diameter Tissue-Engineering Vascular Vessels by Our Novel Integrally Designed Pulsatile Perfusion-Based Bioreactor

et al. (2012) Successful Development of Small Diameter Tissue-Engineering Vascular Vessels by Our Novel Integrally Designed Pulsatile Perfusion-Based Bioreactor. PLoS ONE 7(8): e42569. doi:10.1371/journal.pone.0042569 Successful Development of Small Diameter Tissue- Engineering Vascular Vessels by Our Novel Integrally Designed Pulsatile Perfusion-Based Bioreactor Lei Song 0 Qiang Zhou 0 Ping Duan 0 Ping Guo 0 Dianwei Li 0 Yuan Xu 0 Songtao Li 0 Fei Luo 0 Zehua Zhang 0 Mitsunobu R. Kano, Okayama University, Japan 0 Department of Orthopaedics, First Affiliated Hospital, Third Military Medical University , Chongqing , People's Republic of China Small-diameter (,4 mm) vascular constructs are urgently needed for patients requiring replacement of their peripheral vessels. However, successful development of constructs remains a significant challenge. In this study, we successfully developed small-diameter vascular constructs with high patency using our integrally designed computer-controlled bioreactor system. This computer-controlled bioreactor system can confer physiological mechanical stimuli and fluid flow similar to physiological stimuli to the cultured grafts. The medium circulating system optimizes the culture conditions by maintaining fixed concentration of O2 and CO2 in the medium flow and constant delivery of nutrients and waste metabolites, as well as eliminates the complicated replacement of culture medium in traditional vascular tissue engineering. Biochemical and mechanical assay of newly developed grafts confirm the feasibility of the bioreactor system for smalldiameter vascular engineering. Furthermore, the computer-controlled bioreactor is superior for cultured cell proliferation compared with the traditional non-computer-controlled bioreactor. Specifically, our novel bioreactor system may be a potential alternative for tissue engineering of large-scale small-diameter vascular vessels for clinical use. - Funding: This work is supported by the National High Technology Research and Development Program (863) (No. 2006AA02Z4E3) and the National Natural Science Foundation of China (No. 81027005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Peripheral vascular disease becomes an increasing health and socio-economic burden in most countries. Surgical bypass with autologous vessels remains the main treatment nowadays. However, usable vessels are not often available due to vascular disease, amputation, as well as previous harvest [1]. Thus, smalldiameter (,4 mm) vascular substitutes are urgently needed for patients requiring replacement of their peripheral vessels. The most common alternative to autologous grafts is the use of synthetic small-diameter vascular grafts made of materials such as Dacron or expanded polytetrafluoroethylene [2]. However, the small-diameter arterials of low flow pose a different set of design criteria and introduce various problems including thrombogenicity not encountered in large-caliber vascular substitutes where these synthetic vascular grafts have succeed [3]. As a result, tissue engineering emerges as a promising approach for development of small-diameter vascular grafts. The main objective is to generate biological substitutes of small-diameter arterial conduits with functional characteristics of native vessels with cellular components. Many challenges exist in the tissue engineering of smalldiameter vascular grafts. The greatest one is the development of functional grafts with high patency as well as the antithrombotic properties [4]. Furthermore, it will be meeting all the criteria for tissue engineering of functional large-diameter vascular grafts which should possess mechanical properties such as high burst strength and simultaneous high compliance. In addition, there still exists a barrier to widespread application and low-cost mass production. Current strategies for vascular tissue engineering use arterial wall cells including endothelial cells (ECs) and smooth muscle cells (SMCs) with or without a biodegradable scaffold. Bioreactors technology and bioprocess engineering principles, which can impart physiologically similar biochemical and mechanical stimuli to engineered grafts, are well accepted to facilitate the aseptic growth and maturation of functional grafts [5]. These physiologically similar stimuli are necessary for development of functional vascular grafts, as ECs and SMCs are significantly influenced by local fluid dynamics. Both shear stress and stretch stress are vital to growth and maturation of ECs and SMCs [6,7]. Perfusion-based culture bioreactors have many advantages in vascular tissue engineering, as complex chemical and mechanical stimuli essential to appropriate development can be better achieved in a controllable manner than traditional static cell culture systems [8]. Many perfusion-based devices have been developed in which simple mechanical stimuli could be applied simultaneously, however, these systems provide only a limited ranges of mechanical conditions. Specifically, though previous studies have made great efforts in development of bioreactors to produce small-diameter vascular grafts, there still have not been adequate data of functional vascular characters to confirm the feasibility of these bioreactor systems. In the present study, we successfully develop an integrally designed vascular bioreactor system. With the computer-controlled manipulation, this system allows precise adjustment of physiological pressure, and is capable of developing functional small-diameter vascular vessels with high patency as well as antithrombotic properties. Besides, this integrally designed computer-controlled bioreactor system may make industrial large-scale production of functional small-diameter grafts come true. Materials and Methods Reagents were from Sigma (St. Louis, MO) unless stated otherwise. System Setup The newly developed integrated vascular bioreactor system is shown in Fig. 1. Fig. 2 displays the schema of the perfusion-based bioreactor systems. The major components of the culture system include a culture chamber made of plexiglass, a linear motordriven pump, and medium circulating system, a control system, and integrated auxiliary devices including a laminar flow hood, a temperature controller, and an ozonizer. All devices are assembled in an enclosure. This bioreactor system exerting both compression and medium perfusion is machined from Acrylic organic glass. All screw caps are fitted with a silicone rubber O-ring (Jinan Medical Silicone Rubber; Jinan, China) to seal hermetically. Inlet and outlet ports as well as other fluid ports in this system are standard Luer. Holes of 2 mm in diameter are machined in the upper and basal loading plates for the medium to percolate through. The medium circulating system include a mediu (...truncated)