Xiaofeng Cui

HUMAN MICROVASCULATURE FABRICATION USING THERMAL INKJET PRINTING TECHNOLOGY

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.

Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells.

Thermal inkjet printing technology has been applied successfully to cell printing. However, there are concerns that printing process may cause cell damages or death. We conducted a comprehensive study of thermal inkjet printed Chinese hamster ovary (CHO) cells by evaluating cell viability and apoptosis, and possible cell membrane damages. Additionally, we studied the cell concentration of bio‐ink and found optimum printing of concentrations around 8 million cells per mL. Printed cell viability was 89% and only 3.5% apoptotic cells were observed after printing. Transient pores were developed in the cell membrane of printed cells. Cells were able to repair these pores within 2 h after printing. Green fluorescent protein (GFP) DNA plasmids were delivered to CHO‐S cells by co‐printing. The transfection efficiency is above 30%. We conclude that thermal inkjet printing technology can be used for precise cell seeding with minor effects and damages to the printed mammalian cells. The printing process causes transient pores in cell membranes, a process which has promising applications for gene and macroparticles delivery to induce the biocompatibility or growth of engineered tissues. Biotechnol. Bioeng. 2010;106: 963–969.

Thermal inkjet printing in tissue engineering and regenerative medicine.

With the advantages of high throughput, digital control, and highly accurate placement of cells and biomaterial scaffold to the desired 2D and 3D locations, bioprinting has great potential to develop promising approaches in translational medicine and organ replacement. The most recent advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review. Bioprinting has no or little side effect to the printed mammalian cells and it can conveniently combine with gene transfection or drug delivery to the ejected living systems during the precise placement for tissue construction. With layer-by-layer assembly, 3D tissues with complex structures can be printed using scanned CT or MRI images. Vascular or nerve systems can be enabled simultaneously during the organ construction with digital control. Therefore, bioprinting is the only solution to solve this critical issue in thick and complex tissues fabrication with vascular system. Collectively, bioprinting based on thermal inkjet has great potential and broad applications in tissue engineering and regenerative medicine. This review article introduces some important patents related to bioprinting of living systems and the applications of bioprinting in tissue engineering field.

Synergistic action of fibroblast growth factor-2 and transforming growth factor-beta1 enhances bioprinted human neocartilage formation

Bioprinting as a promising but unexplored approach for cartilage tissue engineering has the advantages of high throughput, digital control, and highly accurate placement of cells and biomaterial scaffold to the targeted 3D locations with simultaneous polymerization. This study tested feasibility of using bioprinting for cartilage engineering and examined the influence of cell density, growth, and differentiation factors. Human articular chondrocytes were printed at various densities, stimulated transiently with growth factors and subsequently with chondrogenic factors. Samples were cultured for up to 4 weeks to evaluate cell proliferation and viability, mechanical properties, mass swelling ratio, water content, gene expression, ECM production, DNA content, and histology. Bioprinted samples treated with FGF‐2/TGF‐β1 had the best chondrogenic properties among all groups apparently due to synergistic stimulation of cell proliferation and chondrogenic phenotype. ECM production per chondrocyte in low cell density was much higher than that in high cell seeding density. This finding was also verified by mechanical testing and histology. In conclusion, cell seeding density that is feasible for bioprinting also appears optimal for human neocartilage formation when combined with appropriate growth and differentiation factors. Biotechnol. Bioeng. 2012;109: 2357–2368.

Free Fatty Acids-Sensing G Protein-Coupled Receptors in Drug Targeting and Therapeutics

G protein-coupled receptor (GPCR) (also known as seven-transmembrane domain receptor) superfamily represents the largest protein family in the human genome. These receptors respond to various physiological ligands such as photons, odors, pheromones, hormones, ions, and small molecules including amines, amino acids to large peptides and steroids. Thus, GPCRs are involved in many diseases and the target of around half of all conventional drugs. The physiological roles of free fatty acids (FFAs), in particular, long-chain FFAs, are important for the development of many metabolic disease including obesity, diabetes, and atherosclerosis. In the past half decade, deorphanization of several GPCRs has revealed that GPR40, GPR41, GPR43, GPR84 and GPR120 sense concentration of extracellular FFAs with various carbon chain lengths. GPR40 and GPR120 are activated by medium- and long-chain FFAs. GPR84 is activated by medium- chain, but not long-chain, FFAs. GPR41 and GPR43 are activated by short-chain FFAs. GPR40 is highly expressed in pancreatic beta cells and plays a crucial role in FFAs-induced insulin secretion. GPR120 is mainly expressed in enteroendocrine cells and plays an important role for FFAs-induced glucagon-like peptide-1. GPR43 is abundant in leukocytes and adipose tissue, whilst GPR41 is highly expressed in adipose tissue, the pancreas and leukocytes. GPR84 is expressed in leukocytes and monocyte/macrophage. This review aims to shed light on the physiological roles and development of drugs targeting these receptors.

Loading dependent swelling and release properties of novel biodegradable, elastic and environmental stimuli-sensitive polyurethanes

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.

Structured three-dimensional co-culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy

Menisci play a crucial role in weight distribution, load bearing, shock absorption, lubrication, and nutrition of articular cartilage within the knee joint. Damage to the meniscus typically does not heal spontaneously due to its partial avascular nature. Partial or complete meniscectomy is a common clinical treatment of the defective meniscus. However, this procedure ultimately leads to osteoarthritis due to increased mechanical stress to the articular cartilage. Meniscus tissue engineering offers a promising solution for partial or complete meniscus deficiency. Mesenchymal stem cells (MSC) have the potential to differentiate into meniscal fibrochondrocyte as well as deliver trophic effects to the differentiated cells. This study tested the feasibility of using MSC co‐cultured with mature meniscal cells (MC) for meniscus tissue engineering. Structured cell pellets were created using MC and MSC at varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) and cultured with or without transforming growth factor‐beta 3 supplemented chondrogenic media for 21 days. The meniscal and hypertrophic gene expression, gross appearance and structure of the pellets, meniscus extracellular matrix (ECM), histology and immunohistochemistry of proteoglycan and collagen were evaluated. Co‐culture of MC with MSC at 75:25 demonstrated highest levels of collagen type I and glycosaminoglycans (GAG) production, as well as the lowest levels of hypertrophic genes, such as COL10A1 and MMP13. All co‐culture conditions showed better meniscus ECM production and hypertrophic inhibition as compared to MSC culture alone. The collagen fiber bundles observed in the co‐cultures are important to produce heterogenic ECM structure of meniscus. In conclusion, co‐culturing MC and MSC is a feasible and efficient approach to engineer meniscus tissue with enhanced ECM production without hypertrophy. Biotechnol. Bioeng. 2012;109: 2369–2380.

Collagen Matrix Alignment Using Inkjet Printer Technology

Collagen fiber orientation plays an important role in many cell properties and actions in vivo. While it is easy to replicate randomly oriented collagen in vitro, it is much more difficult to create aligned collagen matrices for cell culture. In this work, a novel inkjet printer-based collagen alignment technique was established. A collagen type I solution was printed in a line pattern onto glass substrates using a modified inkjet printer. Staining studies indicated that the heat involved in the printing process is not great enough to denature the collagen. The extent of alignment was observed using light microscopy, atomic force microscopy (AFM), and polarized light microscopy. Additionally, cardiomyocytes, which require extracellular matrix alignment to maintain their in vivo phenotype, were cultured on the aligned matrices. The cells grew along the lines of collagen and coordinated beating, indicating the success of the aligned matrix. This collagen alignment technique is cheap, fast, precise, and easy to use in comparison to other current techniques. It may be used to align other extracellular matrix proteins and could even be used to create a three dimensional construct with varying fiber orientations.

Precision Printing of Cells and Biomaterials Onto 3D Matrices

The long term goal of our work is to develop a fabrication technique that allows precision placement of cells inside biomaterial constructs. Such spatial and temporal control of the chemistry and pattern geometry can provide new insights into fundamental aspects of cell-surface interactions. For example, cellular development can be dramatically effected by constraining cells to spread over a specific cell-surface contact area. The cell and biomaterial printing techniques developed here may prove particularly useful for exploring the interactions of anchorage-dependent cells with their environment in vitro. Our recent studies addressed the simultaneous printing of endothelial cells and biomaterials. The studies further demonstrated that cells can be printed onto polymer scaffolds or fibers without significant loss of cell activity. A combination of these methods may result in the construction of vascularized tissue with mechanical properties approaching those of native tissue.© 2007 ASME