Elsevier

Biomaterials

Volume 30, Issue 31, October 2009, Pages 6221-6227
Biomaterials

Human microvasculature fabrication using thermal inkjet printing technology

https://doi.org/10.1016/j.biomaterials.2009.07.056Get rights and content

Abstract

The current tissue engineering paradigm is that successfully engineered thick tissues must include vasculature. As biological approaches alone, such as VEGF, have fallen short of their promises, one may look for an engineering approach to build microvasculature. Layer-by-layer approaches for customized fabrication of cell/scaffold constructs have shown some potential in building complex 3D structures. With the advent of cell printing, one may be able to build precise human microvasculature with suitable bio-ink. Human microvascular endothelial cells (HMVEC) and fibrin were studied as bio-ink for microvasculature construction. Endothelial cells are the only cells to compose the human capillaries and also form the entire inner lining of cardiovascular system. Fibrin has been already widely recognized as tissue engineering scaffold for vasculature and other cells, including skeleton/smooth muscle cells and chondrocytes. In our study, we precisely fabricated micron-sized fibrin channels using a drop-on-demand polymerization. This printing technique uses aqueous processes that have been shown to induce little, if any, damage to cells. When printing HMVEC cells in conjunction with the fibrin, we found the cells aligned themselves inside the channels and proliferated to form confluent linings. The 3D tubular structure was also found in the printed patterns. We conclude that a combined simultaneous cell and scaffold printing can promote HMVEC proliferation and microvasculature formation.

Introduction

The goal of tissue engineering is to solve the organ donor shortage by fabricating the replacement for the lost or damaged tissues and organs [1]. Recently, there are many successes achieved in tissue engineering. However, these successes are limited to relatively thin tissue structures, like skin and bladder [2], [3]. These engineered tissues can be supported by the diffusion of nutrients from the host vasculature. However, when the thickness of the engineered tissue exceeds to 150–200 μm, it will surpass the oxygen diffusion limitation. Therefore, tissue engineers must create functional vasculatures into the engineered tissues to supply the cells with oxygen and nutrients, and to remove the waste products from the cells [4]. This is an unsolved issue in traditional tissue engineering [5]. This critical issue could be solved by cell printing technology, which is based on inkjet printing.

Inkjet printing is a non-contact printing technique. Inkjet printers have the ability to reproduce the data onto substrate with tiny ink drops by receiving data from computers [6]. Drop-on-demand means the ink drops are ejected only where and when they are required to create the images on the substrate. The inkjet printer has high operating frequency, high orifice density, integrated power, and interconnected electronics. In thermal inkjet printers, small air bubbles are created by heating and then collapse to provide the pressure pulse to eject a very tiny drop of ink out of the nozzle [6]. The current pulse lasts a few microseconds and raises the plate temperature as high as 300 °C [7]. Inkjet printing technology has already been widely used in electronics and micro-engineering industries for printing electronic materials and complex integrated circuits [8]. Recently, inkjet technology has also been successfully applied in biomedical field [9]. Although biological molecules and structures are usually assumed to be fragile and sensitive, DNA molecules have been directly printed onto glass slides using commercial available inkjet printers for high-density DNA microarray fabrication [10].

Our lab has successfully developed a novel inkjet printing application using the commercially available inkjet printers to print cells and biomaterials for 3D cellular scaffolds [11]. We showed that the standard HP and Canon desktop inkjet printers can be modified to perform cell printing. Organ printing, defined as computer-aided inkjet based tissue engineering, has the advantage to construct 3D structures with living biological elements. An important advantage of this process is the ability to simultaneously deposit living cells, nutrients, growth factors, therapeutic drugs along with biomaterial scaffolds at the right time and location [12], [13], [14]. This technology can also be used for the microvasculature fabrication using appropriate biomaterials and cells.

Fibrin plays a significant role in natural wound healing. Fibrin gel has been widely used as sealant and adhesive during surgery. Fibrin glue is used as skin graft and tissue-engineered skin replacement [15]. Fibrin can be produced from the patients' own blood and used as an autologous scaffold for tissue engineering [16]. Fibrin is polymerized using fibrinogen and thrombin solutions at room temperature [17]. Fibrin gels might promote cell migration, proliferation, and matrix synthesis through the incorporation of the transforming growth factor β and platelet derived growth factors [18]. Fibrin has also been utilized in tissue engineering to engineer tissues with skeletal muscle cells [19], smooth muscle cells [20], and chondrocytes [21].

Endothelial cells form the inner lining of the whole cardiovascular system and have a remarkable capacity to adjust their number and arrangement to suit local requirements. Almost all tissues depend on a blood supply and the blood supply depends on endothelial cells. Endothelial cells are the only cells to form capillaries. They create an adaptable life-support system spreading into almost every region of the body. Endothelial cells extending and remodeling the network of blood vessels make it possible for tissue growth and repair (angiogenesis) [22].

In our study, a modified Hewlett–Packard Deskjet 500 thermal inkjet printer was used to simultaneously deposit human microvascular endothelial cells and fibrin to form the microvasculature. HP Deskjet 500 inkjet printer has a droplet volume of 130 pL. There are 50 firing nozzles on the printer head and the actual heating occurs in a 10 -μs pulse. The energy supplied during the printing process is transferred into kinetic energy and heating of the ink drop. Mathematical modeling studies indicated that the bulk drop temperature in the ink rises between 4 and 10 degrees above ambient during printing. This makes it possible for printing living systems [23]. It has been proved successful to print cell suspensions [24].

Section snippets

Materials

Human microvascular endothelial cells (HMVEC) were kindly provided by Professor Peter I. Lelkes at Drexel University. MCDB 131 medium, fetal bovine serum, penicillin and streptomycin, sodium bicarbonate, l-glutamine, hydrocortisone, human recombinant epidermal growth factor, heparin, Dulbecco's phosphate buffered saline solution (DPBS), trypsin–EDTA, fibrinogen from bovine plasma, thrombin from bovine plasma were from Sigma Chemicals (St. Louis, MO, USA). Live/Dead Viability/Cytotoxicity Kit

Osmolality of thrombin and fibrinogen solution

Printed fibrin scaffold with minimum thrombin diffusion and scaffold deformations were obtained using the concentrations of 50 unit/ml thrombin, 80 mm CaCl2, and 60 mg/ml fibrinogen (Table 1). Osmolality of thrombin solution of 50 unit/ml thrombin and 80 mm Ca2+ in 1× DPBS was 359 mOsm. Osmolality of 60 mg/ml fibrinogen in distilled water was 341 mOsm. The osmolality of MCDB 131 culture media was 348 mOsm. The osmolalities of these solutions were very close. After fibrin scaffold was printed, only minor

Conclusions

From the printing study of the fibrin gel and HMVEC for microvasculature fabrication, we conclude that human microvascular endothelial cells can be simultaneously deposited along with the appropriate biomaterials (fibrin) for microvasculature fabrication using the modified thermal inkjet printers. The printed endothelial cells proliferate to form a confluent lining along with the fibrin scaffold after 21 days of culture. The rendered 3D channel structure through the series confocal images at

Acknowledgements

The authors would like to acknowledge Dr. Andrew Mount, Dr. Sahil Jalota, Dr. Neeraj, Gohad, Dr. Jake Isenburg, and Ms. Cassie Gregory for suggestions and technical support, Mr. Jim Roberts for manuscript proofreading. This work was partially founded by NSF grant # EEC 0609035 and NSF EFRI.

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