Cartilage and bone tissue engineering using adipose stromal/stem cells spheroids as building blocks

Scaffold-free techniques in the developmental tissue engineering area are designed to mimic in vivo embryonic processes with the aim of biofabricating, in vitro, tissues with more authentic properties. Cell clusters called spheroids are the basis for scaffold-free tissue engineering. In this review, we explore the use of spheroids from adult mesenchymal stem/stromal cells as a model in the developmental engineering area in order to mimic the developmental stages of cartilage and bone tissues. Spheroids from adult mesenchymal stromal/stem cells lineages recapitulate crucial events in bone and cartilage formation during embryogenesis, and are capable of spontaneously fusing to other spheroids, making them ideal building blocks for bone and cartilage tissue engineering. Here, we discuss data from ours and other labs on the use of adipose stromal/stem cell spheroids in chondrogenesis and osteogenesis in vitro. Overall, recent studies support the notion that spheroids are ideal "building blocks" for tissue engineering by “bottom-up” approaches, which are based on tissue assembly by advanced techniques such as three-dimensional bioprinting. Further studies on the cellular and molecular mechanisms that orchestrate spheroid fusion are now crucial to support continued development of bottom-up tissue engineering approaches such as three-dimensional bioprinting.     

Effects of flow geometry on blood viscoelasticity

The viscoelastic properties of blood are dominated by microstructures formed by red cells. The microstructures are of several types such as irregular aggregates, rouleaux, and layers of aligned cells. The dynamic deformability of the red cells, aggregation tendency, cell concentration, size of confining vessel and rate of flow are determining factors in the microstructure. Viscoelastic properties, viscosity and elasticity, relate to energy loss and storage in flowing blood while relaxation time and Weissenberg number play a role in assessing the importance of the elasticity relative to the viscosity. These effects are shown herein for flow in a large straight cylindrical tube, a small tube, and a porous medium. These cases approximate the geometries of the arterial system: large vessels, small vessels and vessels with many branches and bifurcations. In each case the viscosity, elasticity, relaxation time and Weissenberg number for normal human blood as well as blood with enhanced cell aggregation tendency and diminished cell deformability are given. In the smaller spaces of the microtubes and porous media, the diminished viscosity shows the possible influence of the F?hraeus-Lindqvist effect and at high shear rates, the viscoelasticity of blood shows dilatancy. This is true for normal, aggregation enhanced and hardened cells.     

Scaffold‐free inkjet printing of three‐dimensional zigzag cellular tubes

The capability to print three‐dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform‐assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)‐based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post‐printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72‐h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold‐free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof‐of‐concept study, the resulting fabrication knowledge helps print tissue‐engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152–3160. © 2012 Wiley Periodicals, Inc.     

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.     

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.     

Essential steps in bioprinting: From pre- to post-bioprinting

An increasing demand for directed assembly of biomaterials has inspired the development of bioprinting, which facilitates the assembling of both cellular and acellular inks into well-arranged three-dimensional (3D) structures for tissue fabrication. Although great advances have been achieved in the recent decade, there still exist issues to be addressed. Herein, a review has been systematically performed to discuss the considerations in the entire procedure of bioprinting. Though bioprinting is advancing at a rapid pace, it is seen that the whole process of obtaining tissue constructs from this technique involves multiple-stages, cutting across various technology domains. These stages can be divided into three broad categories: pre-bioprinting, bioprinting and post-bioprinting. Each stage can influence others and has a bearing on the performance of fabricated constructs. For example, in pre-bioprinting, tissue biopsy and cell expansion techniques are essential to ensure a large number of cells are available for mass organ production. Similarly, medical imaging is needed to provide high resolution designs, which can be faithfully bioprinted. In the bioprinting stage, compatibility of biomaterials is needed to be matched with solidification kinetics to ensure constructs with high cell viability and fidelity are obtained. On the other hand, there is a need to develop bioprinters, which have high degrees of freedom of movement, perform without failure concerns for several hours and are compact, and affordable. Finally, maturation of bioprinted cells are governed by conditions provided during the post-bioprinting process. This review, for the first time, puts all the bioprinting stages in perspective of the whole process of bioprinting, and analyzes their current state-of-the art. It is concluded that bioprinting community will recognize the relative importance and optimize the parameter of each stage to obtain the desired outcomes.     

In vivo evaluation of bioprinted prevascularized bone tissue

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.     

Three-dimensional bioprinting in tissue engineering and regenerative medicine

With the advances of stem cell research, development of intelligent biomaterials and three-dimensional biofabrication strategies, highly mimicked tissue or organs can be engineered. Among all the biofabrication approaches, bioprinting based on inkjet printing technology has the promises to deliver and create biomimicked tissue with high throughput, digital control, and the capacity of single cell manipulation. Therefore, this enabling technology has great potential in regenerative medicine and translational applications. The most current advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review, including vasculature, muscle, cartilage, and bone. In addition, the benign side effect of bioprinting to the printed mammalian cells can be utilized for gene or drug delivery, which can be achieved conveniently during precise cell placement for tissue construction. With layer-by-layer assembly, three-dimensional tissues with complex structures can be printed using converted medical images. Therefore, bioprinting based on thermal inkjet is so far the most optimal solution to engineer vascular system to the thick and complex tissues. Collectively, bioprinting has great potential and broad applications in tissue engineering and regenerative medicine. The future advances of bioprinting include the integration of different printing mechanisms to engineer biphasic or triphasic tissues with optimized scaffolds and further understanding of stem cell biology.     

细胞打印技术及应用

细胞打印技术是一种在体外构造具有生物活性的三维多细胞体系的先进技术。近年来,有关细胞打印技术的研究引起广泛的关注,其原因在于该领域具有明显的学科交叉与渗透融合的特点,它处于生命科学与快速成型技术、生物制造技术、生物科学和材料科学的交汇点。更加值得关注的是它为组织工程学突破二维研究的局限性,在三维尺度上精确控制与人体组织或器官相似的三维构造体方面的研究提供了一种新的思路。基于这一技术不仅在三维组织工程,还对细胞生物学、高通量药物筛选及细胞传感器等方面的前沿问题均有广阔的研究应用前景,介绍了近年来开发用于细胞打印的技术及其潜在的应用。     

ECM concentration and cell-mediated traction forces play a role in vascular network assembly in 3D bioprinted tissue

Tissue vascularization is critical to enable oxygen and nutrient supply. Therefore, establishing expedient vasculature is necessary for the survival of tissue after transplantation. The use of biomechanical forces, such as cell-induced traction forces, may be a promising method to encourage growth of the vascular network. Three-dimensional (3D) bioprinting, which offers unprecedented versatility through precise control over spatial distribution and structure of tissue constructs, can be used to generate capillary-like structures in vitro that would mimic microvessels. This study aimed to develop an in vitro, 3D bioprinted tissue model to study the effect of cellular forces on the spatial organization of vascular structures and tissue maturation. The developed in vitro model consists of a 3D bioprinted polycaprolactone (PCL) frame with a gelatin spacer hydrogel layer and a gelatin–fibrin–hyaluronic acid hydrogel layer containing normal human dermal fibroblasts and human umbilical vein endothelial cells printed as vessel lines on top. The formation of vessel-like networks and vessel lumens in the 3D bioprinted in vitro model was assessed at different fibrinogen concentrations with and without inhibitors of cell-mediated traction forces. Constructs containing 5 mg/ml fibrinogen had longer vessels compared to the other concentrations of fibrinogen used. Also, for all concentrations of fibrinogen used, most of the vessel-like structures grew parallel to the direction the PCL frame-mediated tensile forces, with very few branching structures observed. Treatment of the 3D bioprinted constructs with traction inhibitors resulted in a significant reduction in length of vessel-like networks. The 3D bioprinted constructs also had better lumen formation, increased collagen deposition, more elaborate actin networks, and well-aligned matrix fibers due to the increased cell-mediated traction forces present compared to the non-anchored, floating control constructs. This study showed that cell traction forces from the actomyosin complex are critical for vascular network assembly in 3D bioprinted tissue. Strategies involving the use of cell-mediated traction forces may be promising for the development of bioprinting approaches for fabrication of vascularized tissue constructs.     

Windows of operation for bioreactor design for the controlled formation of tissue‐engineered arteries

The availability of large numbers of units of artificial arteries would offer significant benefits to the clinical management of bypass surgery. Tissue engineering offers the potential of providing vessels that can mimic the morphology, function, and physiological environment of native vessels. Ideally this would involve culturing stem cells in vitro within a biodegradable tubular scaffold so as to construct tissue for implantation. Essential to establishing a robust process for the production of tissue‐engineered arteries is the understanding of the impact of changes in the operating conditions and bioreactor design on the construct formation. In this article, models of transport phenomena were developed to predict the critical flow rates and mass transfer requirements of a prototype bioreactor for the formation of tissue‐engineered arteries. The impact of the cell concentration, tube geometry, oxygen effective diffusivity in alginate, substrate and metabolite concentration levels, feed rate, and recycle rate on the design of the bioreactor was visualized using windows of operation and contour plots. The result of this analysis determined the best configuration of the bioreactor that meets the cellular transport requirements as well as being reliable in performance while seeking to reduce the amount of nutrients to be used. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

3D bioprinting and the current applications in tissue engineering

Bioprinting as an enabling technology for tissue engineering possesses the promises to fabricate highly mimicked tissue or organs with digital control. As one of the biofabrication approaches, bioprinting has the advantages of high throughput and precise control of both scaffold and cells. Therefore, this technology is not only ideal for translational medicine but also for basic research applications. Bioprinting has already been widely applied to construct functional tissues such as vasculature, muscle, cartilage, and bone. In this review, the authors introduce the most popular techniques currently applied in bioprinting, as well as the various bioprinting processes. In addition, the composition of bioink including scaffolds and cells are described. Furthermore, the most current applications in organ and tissue bioprinting are introduced. The authors also discuss the challenges we are currently facing and the great potential of bioprinting. This technology has the capacity not only in complex tissue structure fabrication based on the converted medical images, but also as an efficient tool for drug discovery and preclinical testing. One of the most promising future advances of bioprinting is to develop a standard medical device with the capacity of treating patients directly on the repairing site, which requires the development of automation and robotic technology, as well as our further understanding of biomaterials and stem cell biology to integrate various printing mechanisms for multi‐phasic tissue engineering.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

Morphology of central compartment and vasculature of the gills of Lepidosiren paradoxa (Fitzinger)

The four paired gill arches of the South American lungfish Lepidosiren paradoxa contain single branchial arteries directly connecting dorsal and ventral arteries. In gill arches 3 and 4 the branchial arteries also supply looped arlerioles and capillaries to much-reduced gill filaments. Regulation of blood between these routes is thought to be by alteration of vascular resistance. Within the filaments, extensive subepithelial capillary networks and numerous small pumps connect lymphatic vessels in the central connective tissue compartment with venules which, in turn, drain to paired branchial veins.
The features of the endothelium of many of the filament blood vessels suggest extensive transporting, haematolytic and granulopoeitic functions. Large numbers of macrophages pack the connective tissue. Many contain extensive quantities of haemosiderin.     

Method of quantitative assessment of the cerebral vascular bed in experimental morphologic studies

Cleared brain sections 20 mkm thick with injected vessels are photographed and then projected on the screen. Vascular contours are sketched on the paper, cut out and weighed. Vessel-capillary network/the whole section field ratio in percents is then calculated. Having separated the area of the vessels, the area occupied by the capillaries is divided into the mean capillary diameter, thus it is possible to estimate the capillary length per area unit and the volume of the brain substance.     

脱细胞基质生物墨水制备方法及应用进展

组织器官脱细胞后制备成的脱细胞基质 (Decellularized extracellular matrix,dECM) 含有许多蛋白质和生长因子,不仅能够为细胞提供三维支架还能够调控细胞再生,是目前最具有生物结构的生物材料。3D生物打印可以层层打印dECM和自体细胞的组合,构建载细胞组织结构。文中综述了不同来源的组织器官脱细胞基质生物墨水制备方法,包括脱细胞、交联等,以及脱细胞基质生物墨水在生物打印中的应用,并展望了其未来的应用前景。     

Structural and histochemical aspects of perirenal adipose tissue in fetal pigs: relationships between stromal-vascular characteristics and fat cell concentration and enzyme activity

Samples of perirenal adipose tissue were obtained from four fetuses from each of seven crossbred gilts at each of three stages of gestation: 70, 90, and 110 days. Samples were routinely prepared for histochemistry and histology. At each age, the largest fat cell clusters were consistently located near points where large blood vessels entered the loose connective tissue. Cell-cluster size decreased with distance from the entry points of large blood vessels. Fat cells proximal to entry points of large arterioles and fat cells distal to entry points of large arterioles were the same size. Enzyme cytochemistry disclosed that reactions for glucose-6-phosphate dehydrogenase (G6PDH), lipoprotein lipase (LPL) and NADH-TR enzymes were reduced in distal (relative to entry points of large arterioles) adipocytes compared with proximal adipocytes. Reactions for succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) in adipocytes were not influenced by location within the tissue. Small fat cell clusters with sparse capillary beds surround arterioles in distal areas of sections from fetuses at 70, 90, and 110 days of gestation. In the proximal areas of sections from 110-day-old fetuses, arterioles were surrounded by large fat cell clusters with dense capillary beds. These characteristics serve to distinguish perirenal depots from subcutaneous depots in the fetus.     

Ultrastructure and functional versatility of hirudinean botryoidal tissue

In leeches, the botryoidal tissue is composed of two different cell types--granular botryoidal cells and flattened endothelial-like cells--localized in the loose connective tissue between the gut and the body wall sac. We have observed that the botryoidal tissue undergoes functional and structural modifications in response to the different needs arising during the life-cycle of the animal. In healthy, untreated leeches, botryoidal cells are organized in cords or clusters, sometimes surrounding few, small lacunae. Conversely, in wounded animals we have observed the transition of the botryoidal tissue from cluster/cord-like structures to a hollow/tubular architecture, typical of pre-vascular structures. We have documented in botryoidal cell cytoplasm the presence of large calcium storage. Moreover, the cytoplasm of botryoidal cells was filled with granules of different form and size, containing iron or melanin, as tested by classic histochemical methods. The presence of elements like iron and calcium was confirmed by the well-established EDS analysis. In response to a surgical wound, botryoidal tissue cells changed their shape and formed new capillary vessels. Concurrently, botryoidal cells secreted iron from cytoplasmic granules into the new cavity: this secretory activity appeared to be related to intracellular calcium fluctuations. At the end of the angiogenic process, botryoidal cells lost their contact with the basal lamina and moved freely in the circulating fluid towards the lesioned area. Interestingly, circulating botryoidal cells were found to carry melanin in the wounded area. This function is probably involved in defense processes. Thus, we have shown that stimulated botryoidal tissue displays a variety of striking structural, secretory and defensive activities.     

Ultrastructural and morphometric studies related to expression of the cell adhesion molecule PECAM-1/CD31 in developing rat lung.

It has recently been postulated that platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) might play a role in vascular tube formation. To evaluate the role of PECAM-1/CD31 in the formation of the capillary network in vivo, we conducted an ultrastructural immunohistochemical evaluation of the localization of PECAM-1/CD31 and its developmentally regulated expression in the periphery of the lungs of fetal, newborn, and adult rats. PECAM-1/CD31 was present mainly on luminal surfaces and at the junctions between endothelial cells. Moreover, in fetal lung, products of the immunoreaction were also found on the abluminal surfaces of endothelial cells. To relate those findings to the developmental changes in the capillary area of the lung, we performed a morphometric study of electronmicrographs. The cross-sectional area of blood vessels at the periphery of the lungs was significantly greater in 15-19-day-old fetuses than in postpartum animals (p<0.0001). Disappearance of the expression of PECAM-1/CD31 on the abluminal endothelial surface paralleled the changes in the cross-sectional area of blood vessels that occurred during the perinatal period. (J Histochem Cytochem 48:1283-1289, 2000)     

Plug Effect of Erythrocytes in Capillary Blood Vessels

As an idealized problem of the motion of blood in small capillary blood vessels, the low Reynolds number flow of plasma (a newtonian fluid) in a circular cylindrical tube involving a series of circular disks is studied. It is assumed in this study that the suspended disks are equally spaced along the axis of the tube, and that their centers remain on the axis of the tube and that their faces are perpendicular to the tube axis. The inertial force of the fluid due to the convective acceleration is neglected on the basis of the smallness of the Reynolds number. The solution of the problem is derived for a quasi-steady flow involving infinitesimally thin disks. The numerical calculation is carried out for a set of different combinations of the interdisk distance and the ratio of the disk radius to the tube radius. The ratio of the velocity of the disk to the average velocity of the fluid is calculated. The different rates of transport of red blood cells and of plasma in capillary blood vessels are discussed. The average pressure gradient along the axis of the tube is computed, and the dependence of the effective viscosity of the blood on the hematocrit and the diameter of the capillary vessel is discussed.