Copyright (c) 2009 All rights reserved. http://works.bepress.com/thomas_boland Recent documents in Thomas Boland en-us Thu, 22 Oct 2009 23:19:06 PDT 3600 http://works.bepress.com/thomas_boland/17 http://works.bepress.com/thomas_boland/17 Wed, 21 Oct 2009 10:33:09 PDT Self-assembled monolayers (SAMs) have become a standard tool for exploring surface interactions. Although well characterized, SAMs are known to undergo structural and conformational changes in the presence of solution, yet the ability to quantify these changes remains an obstacle due to limited analytical techniques. In this study, we determine changes in structure and conformation of CH3, OH, and COOH terminated hexadecanethiols on gold in water by means of a new technique known as evanescence reflection spectroscopy. This FTIR application, in conjunction with a semiempirical formalism, is capable of providing both qualitative and quantitative understanding of the molecular structure and orientation at the solid/liquid interface. Laura Pardo Original Publications http://works.bepress.com/thomas_boland/16 http://works.bepress.com/thomas_boland/16 Wed, 21 Oct 2009 10:31:05 PDT Commercial ink-jet printers were used with little modification to deposit alkanethiols onto gold substrata and several proteins onto silica supports. The resulting patterns of alkanethiols form self-assembled layers comparable to those obtained by microcontact printing or solution adsorption. The method has been used successfully to create binary chemical gradients and patterns of tertiary functionality. The proteins form dense patterns on the substrates and seem to maintain their configuration as measured by their ability to bind their specific ligands. Four different proteins were printed simultaneously, allowing for positive and negative controls. This "drop-on-demand" printing method is an inexpensive, flexible alternative to current binary technologies of chemically functionalizing surfaces. Laura F. Pardo Original Publications http://works.bepress.com/thomas_boland/15 http://works.bepress.com/thomas_boland/15 Wed, 21 Oct 2009 10:28:01 PDT Tissue engineering technology promises to solve the organ transplantation crisis. However, assembly of vascularized 3D soft organs remains a big challenge. Organ printing, which we define as computer-aided, jet-based 3D tissue-engineering of living human organs, offers a possible solution. Organ printing involves three sequential steps: pre-processing or development of "blueprints" for organs; processing or actual organ printing; and postprocessing or organ conditioning and accelerated organ maturation. A cell printer that can print gels, single cells and cell aggregates has been developed. Layer-by-layer sequentially placed and solidified thin layers of a thermo-reversible gel could serve as "printing paper". Combination of an engineering approach with the developmental biology concept of embryonic tissue fluidity enables the creation of a new rapid prototyping 3D organ printing technology, which will dramatically accelerate and optimize tissue and organ assembly. Vladimir Mironov Review Papers http://works.bepress.com/thomas_boland/14 http://works.bepress.com/thomas_boland/14 Wed, 21 Oct 2009 10:25:08 PDT We recently developed a cell printer (Wilson and Boland, 2003) that enables us to place cells in positions that mimic their respective positions in organs. However, this technology was limited to the printing of two-dimensional (2D) tissue constructs. Here we describe the use of thermosensitive gels to generate sequential layers for cell printing. The ability to drop cells on previously printed successive layers provides a real opportunity for the realization of three-dimensional (3D) organ printing. Organ printing will allow us to print complex 3D organs with computer-controlled, exact placing of different cell types, by a process that can be completed in several minutes. To demonstrate the feasibility of this novel technology, we showed that cell aggregates can be placed in the sequential layers of 3D gels close enough for fusion to occur. We estimated the optimum minimal thickness of the gel that can be reproducibly generated by dropping the liquid at room temperature onto a heated substrate. Then we generated cell aggregates with the corresponding (to the minimal thickness of the gel) size to ensure a direct contact between printed cell aggregates during sequential printing cycles. Finally, we demonstrated that these closely-placed cell aggregates could fuse in two types of thermosensitive 3D gels. Taken together, these data strongly support the feasibility of the proposed novel organ-printing technology. Thomas Boland Original Publications http://works.bepress.com/thomas_boland/13 http://works.bepress.com/thomas_boland/13 Wed, 21 Oct 2009 10:21:11 PDT We have developed several devices for positioning organic molecules, molecular aggregates, cells, and single-cell organisms onto solid supports. These printers can create stable, functional protein arrays using an inexpensive technology. The cell printer allows us to create cell libraries as well as cellular assemblies that mimic their respective position in organs. The printers are derived from commercially available ink-jet printers that are modified to dispense protein or cell solutions instead of ink. We describe here the modifications to the print heads, and the printer hardware and software that enabled us to adapt the ink-jet printers for the manufacture of cell and protein arrays. The printers have the advantage of being fully automated and computer controlled, and allow for the high-throughput manufacture of protein and cell arrays. Cris Wilson Original Publications http://works.bepress.com/thomas_boland/12 http://works.bepress.com/thomas_boland/12 Wed, 21 Oct 2009 10:18:03 PDT We have developed a method for fabricating bacterial colony arrays and complex patterns using commercially available ink-jet printers. Bacterial colony arrays with a density of 100 colonies/cm(2) were obtained by directly ejecting Escherichia coli (E. coli) onto agar-coated substrates at a rapid arraying speed of 880 spots per second. Adjusting the concentration of bacterial suspensions allowed single colonies of viable bacteria to be obtained. In addition, complex patterns of viable bacteria as well as bacteria density gradients were constructed using desktop printers controlled by a simple software program. Tao Xu Original Publications http://works.bepress.com/thomas_boland/11 http://works.bepress.com/thomas_boland/11 Wed, 21 Oct 2009 10:10:13 PDT The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible. Elisabeth A. Roth Original Publications http://works.bepress.com/thomas_boland/10 http://works.bepress.com/thomas_boland/10 Wed, 21 Oct 2009 10:07:04 PDT One of the most important aspects of tissue engineering is the design of the scaffold providing the mechanical strength and access to nutrients for the new tissue. For customized tissue engineering, it is essential to be able to fabricate three-dimensional scaffolds of various geometric shapes, in order to repair defects caused by accidents, surgery, or birth. Rapid prototyping or solid free-form fabrication (SFF) techniques hold great promise for designing three-dimensional customized scaffolds, yet traditional cell-seeding techniques may not provide enough cell mass for larger constructs. This article presents a novel attempt to fabricate three-dimensional scaffolds, using hydrogels combined with cell encapsulation to fabricate high-density tissue constructs. A commercially available stereolithography technique was applied to fabricate scaffolds using poly(ethylene oxide) and poly(ethylene glycol)dimethacrylate photopolymerizable hydrogels. Mechanical characterization shows the constructs to be comparable with soft tissues in terms of elasticity. High cell viability was achieved and high-density constructs fabricated. Busaina Dhariwala Original Publications http://works.bepress.com/thomas_boland/9 http://works.bepress.com/thomas_boland/9 Wed, 21 Oct 2009 10:04:30 PDT The purpose of this study was to explore the use of a commercial thermal printer to deposit Chinese Hamster Ovary (CHO) and embryonic motoneuron cells into pre-defined patterns. These experiments were undertaken to verify the biocompatibility of thermal inkjet printing of mammalian cells and the ability to assemble them into viable constructs. Using a modified Hewlett Packard (HP) 550C computer printer and an HP 51626a ink cartridge, CHO cells and rat embryonic motoneurons were suspended separately in a concentrated phosphate buffered saline solution (3 x). The cells were subsequently printed as a kind of "ink" onto several "bio-papers" made from soy agar and collagen gel. The appearance of the CHO cells and motoneurons on the bio-papers indicated an healthy cell morphology. Furthermore, the analyses of the CHO cell viability showed that less than 8% of the cells were lysed during printing. These data indicate that mammalian cells can be effectively delivered by a modified thermal inkjet printer onto biological substrates and that they retain their ability to function. The computer-aided inkjet printing of viable mammalian cells holds potential for creating living tissue analogs, and may eventually lead to the construction of engineered human organs. Tao Xu Original Publications http://works.bepress.com/thomas_boland/8 http://works.bepress.com/thomas_boland/8 Wed, 21 Oct 2009 10:01:47 PDT David Varghese Original Publications http://works.bepress.com/thomas_boland/7 http://works.bepress.com/thomas_boland/7 Wed, 21 Oct 2009 09:58:10 PDT Complex cellular patterns and structures were created by automated and direct inkjet printing of primary embryonic hippocampal and cortical neurons. Immunostaining analysis and whole-cell patch-clamp recordings showed that embryonic hippocampal and cortical neurons maintained basic cellular properties and functions, including normal, healthy neuronal phenotypes and electrophysiological characteristics, after being printed through thermal inkjet nozzles. In addition, in this study a new method was developed to create 3D cellular structures: sheets of neural cells were layered on each other (layer-by-layer process) by alternate inkjet printing of NT2 cells and fibrin gels. These results and findings, taken together, show that inkjet printing is rapidly evolving into a digital fabrication method to build functional neural structures that may eventually find applications in neural tissue engineering. Tao Xu Original Publications http://works.bepress.com/thomas_boland/6 http://works.bepress.com/thomas_boland/6 Wed, 21 Oct 2009 09:52:59 PDT ecent advances in organ printing technology for applications relating to medical interventions and organ replacement are described. Organ printing refers to the placement of various cell types into a soft scaffold fabricated according to a computer-aided design template using a single device. Computer aided scaffold topology design has recently gained attention as a viable option to achieve function and mass transport requirements within tissue engineering scaffolds. An exciting advance pioneered in our laboratory is that of simultaneous printing of cells and biomaterials, which allows precise placement of cells and proteins within 3-D hydrogel structures. This advance raises the possibility of spatially controlling not only the scaffold structure, but also the type of tissue that can be grown within the scaffold and the thickness of the tissue as capillaries and vessels could be constructed within the scaffolds. Here we summarize recent advances in printing cells and materials using the same device. Thomas Boland Review Papers http://works.bepress.com/thomas_boland/5 http://works.bepress.com/thomas_boland/5 Wed, 21 Oct 2009 09:49:19 PDT Biodegradable polyurethanes (PUs) were synthesized from methylene di-p-phenyl-diisocyanate (MDI), polycaprolactone diol (PCL-diol) and N,N-bis (2-hydorxyethyl)-2-aminoethane-sulfonic acid (BES), serving as a hard segment, soft segment and chain extender, respectively. MDI was chosen due to its reactivity and wide application in synthesis of biomedical polyurethanes due to its reactivity; PCL-diol was chosen because of its biodegradability; and BES was chosen because it allowed the introduction sulfonic acid groups onto the polymer chains. We evaluated the polyurethanes' degradation rate, mechanical properties, hydrophilicity, antithrombogenecity, and ability to support fibroblast cell attachment and growth by comparing with polymers having a 2,2-(methylimino)diethanol (MIDE) chain extender. Mechanical testing demonstrated that the PU containing BES has tensile strengths of about 17 MPa and elongations up to 400%, about three times the strength and four times the elongation than the MIDE based PUs. The polymers showed decreased in vitro degradation rates, lower glass transition temperature (T(g)) and hydrophilicity possibly due to enhanced microphase separation. Preliminary cytocompatibility studies showed that all the PUs are non-toxic, but PU containing BES exhibited much lower cell attachment and proliferation than the MIDE chain extended polymers. An in vitro platelet adhesion assay showed lower platelet attachment on BES containing PU. Additionally, due to the existence of sulfonic acid groups, the BES extended PU became water soluble in basic condition and insoluble in acidic condition, a phenomenon that is reversible at pH value of 8.7, making this a pH sensitive polymer attractive for bioprinting applications. By adding acetic acid into an inkjet cartridge and printing it onto a PU solution with pH above 8.7, precision fabricated scaffolds can be obtained, suggesting that BES based PUs are promising candidates as synthetic inks used for customizable fabrication of tissue engineering scaffolds. Changhong Zhang Original Publications http://works.bepress.com/thomas_boland/4 http://works.bepress.com/thomas_boland/4 Wed, 21 Oct 2009 09:43:32 PDT A novel degradable, elastic, anionic, and linear polyurethane was synthesized from hexamethylene diisocyanate, polycaprolactone diol, and a bicine chain extender. The chemical structure, mechanical properties, degradation rate, and swelling ratio were characterized by comparing the polymer with a polyurethane containing a 2,2-(methylimino) diethanol chain extender. Due to the incorporation of negatively charged carboxyl side groups, the bicine extended polymers exhibited higher micro-phase separation, better mechanical properties in dry condition, and better sensitivity to environmental stimuli than controls, as demonstrated by its high swelling ratio at elevated pH, lower ionic strength, or higher temperature. The swelling ratio of membranes showed reversible change as the function of pH at 37 degrees C, the membranes becoming fully water soluble at pH above 8.3. Nile blue chloride and lysozyme were selected to study their release from this polymer. The release rates of both compounds were significantly influenced by the pH and ionic strength. The swelling ratios were also influenced by lysozyme loading at low pH. The pH dependent properties were used to fabricate scaffolds by drop-on-demand printing. Bicine extended polyurethanes may be of interest for possible drug delivery applications, customizable scaffold fabrication and other potential biomedical applications. Changhong Zhang Original Publications http://works.bepress.com/thomas_boland/3 http://works.bepress.com/thomas_boland/3 Wed, 21 Oct 2009 09:26:18 PDT Rat embryonic hippocampal neurons were cultured in (1) 3D collagen hydrogels as 'entrapped' evenly distributed cells, (2) at the interface of two collagen layers (sandwich model), and (3) on the surface of collagen coated coverslips (2D model). In the 'entrapment' model the neuronal processes grew out of the plane of the cell body and extended into the collagen matrix, in contrast to the sandwich model where the cells and their processes rarely left the plane in which they were seeded. Hippocampal neurons 'entrapped' in the 3D collagen gel grew the same number, but shorter, processes and exhibited improved survival compared to neurons cultured in the 2D model. There was no difference in the electrophysiological properties of the neurons cultured in the 3D compared to the 2D model except in the resting membrane potential and in the duration of the after-hyperpolarization. Spontaneous postsynaptic currents were recorded in 14- and 21-day-old 3D cultures evidencing functional synapse formation. Our results indicate that the physiological characteristics of 3D neuronal cultures are similar to traditional 2D cultures. However, functional 3D networks of hippocampal neurons will be necessary for multi-level circuit formation, which could be essential for understanding the basis of physiological learning and memory. Tao Xu Original Publications http://works.bepress.com/thomas_boland/2 http://works.bepress.com/thomas_boland/2 Wed, 21 Oct 2009 09:17:23 PDT We report to fabricate functional three-dimensional (3D) tissue constructs by using an inkjet based bio-prototyping method. With the use of the modified inkjet printers, contractile cardiac hybrids that exhibit the forms of the 3D rectangular sheet and even the 'half heart' (with two connected ventricles) have been fabricated by arranging alternate layers of biocompatible alginate hydrogels and mammalian cardiac cells according to pre-designed 3D patterns. In this study, primary feline adult and H1 cardiomyocytes were used as model cardiac cells. Alginate hydrogels with controlled micro-shell structures were built by spraying cross-linkers in micro-drops onto un-gelled alginic acid. The cells remained viable in constructs as thick as 1 cm due to the programmed porosity. Microscopic and macroscopic contractile functions of these cardiomyocytes constructs were observed in vitro. These results suggest that the inkjet bio-prototyping method could be used for hierarchical design of functional cardiac pseudo tissues, balanced with porosity for mass transport and structural support. Tao Xu Original Publications http://works.bepress.com/thomas_boland/1 http://works.bepress.com/thomas_boland/1 Wed, 21 Oct 2009 09:06:43 PDT 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. Xiaofeng Cui Original Publications