Spatial Effects of Stem Cell Engagement in 3D Printing Constructs

Review Article

J Stem Cells Res, Rev & Rep. 2014;1(2): 1007.

Spatial Effects of Stem Cell Engagement in 3D Printing Constructs

Xiaohong Wang1,2*

1Department of Mechanical Engineering, Tsinghua University, P.R. China

2State Key Laboratory of Materials Processing and Die & Mould Technology, Huazhong University of Science and Technology, P.R. China

*Corresponding author: Xiaohong Wang, Department of Mechanical Engineering, Key Laboratory for Advanced Materials Processing Technology, Ministry of Education & Center of Organ Manufacturing, Tsinghua University, Beijing 100084, P.R. China

Received: Aug 20, 2014; Accepted: Aug 23, 2014; Published: Aug 25, 2014


Three-Dimensional Printing (3DP) technology is a remarkable new invention developed for complex organ manufacturing. For the multiple nozzle 3DP techniques, it is capable of placing various cells and Extracellular Matrices (ECMs) into predestinated locations which mimic their respective positions in a living organ. The High-Fidelity (Hi-Fi) constructs hold enormous therapeutic potential in organ transplantation and regenerative medicine. For the one-nozzle 3DP techniques, when one stem cell type is assembled into a spatially organized construct, the predesigned architecture facilitates the stem cells to grow, interact, organize, and differentiate successively with a cocktail combination of growth factor engagement based on their positions within the construct. A series of morphological and functional changes, such as vascularization and angiogenesis, are accompanied during the stem cell differentiation, tissue formation and organ maturation stages. This review mainly focuses on the spatial effects of sequencial differentiation procedures of adipose-derived stem cells in a 3DP construct

Keywords: Stem cells; Gelatin/alginate/fibrin matrices; Three-dimensional printing (3DP); Spatial effects; Inducement/engagement


Organs in vivo have very heterogeneous but well-organized Three-Dimensional (3D) tissues [1]. For example, large blood vessel, serving as blood passage channels, has three lamellar cellbased layers: adventitia, media and intima. The three layers, with thicknesses of about hundred microns, are diversified in terms of cell type, Extracellular Matrix (ECM) composition, and functional properties. To date one of the major obstacles in complex organ manufacturing is to incorporate multiple cell types and maintain heterotypic cell viabilities during tissue formation and structural organization [2-4]. Over the last decade various adult cells, such as hepatocytes, cardiomyocyte, Endothelial Cells (ECs), Smooth Muscle Cells (SMCs), fibroblasts, have been used in tissue/organ regeneration [5]. Developing a 3D construct that incorporates heterotypic cell - cell interactions (e.g. endothelial cell - smooth muscle cell) in a well-controlled manner has been proved to be critical for complex organ manufacturing. The formation of organized and functional heterotypic tissues in a 3D construct has been proved to be a very difficult task.

Three-dimensional printing (3DP), also called Rapid Prototyping (RP) and Additive Manufacturing (AM), consists of a series of novel platforms that can simultaneously assemble cells and Extracellular Matrices (ECMs) from digital models in a precisely controlled layerby- layer fashion, including material composition, architecture, and internal pore size, interconnectivity, branching, geometry and orientation [6-11]. The thickness of each layer can be controlled by the parameters of the printers, nozzle size, material concentration, and extruding speed. The printed 3D constructs proved to maintain the multilayered configurations for cells from microscale to macroscale [12]. This is essential to create 3D biomimetic organs because the layered structure of most tissues varies in terms of cellular composition and extracellular matrix properties. Recently, 3DP techniques have become more and more popular with their fabrication abilities of custom complex organs. However, if onenozzle 3D printer is used and one stem cell type is printed, a cocktail growth factor engagement is necessary to induce the stem cells to differentiate into different target cell types [13,14].

There is increasing experimental evidence that living cells are inherently sensitive to physical, biochemical and chemical stimuli from their surrounding environments [15]. Stem cell differentiation depends on the growth factor inducement and gradients/ concentrations [16]. The local environmental cues or “niches” determine cell-specific recruitment, migration, proliferation, differentiation and the production of the numerous proteins needed for hierarchical tissue organization [17]. Several studies have used stem cell niches to observe the behavior of extending or regenerating axons in response to different in vitro gradients of chemotropic factors [18]. Others built a micro fluidic gradient to study neurite guidance by diffusible factors in a 3D in vitro cell culture model [19]. A growing number of studies report stem cell engagement effects within various natural or synthetic hydrogels [20].

To date, the majority of related studies have used 2D or quasi-3D environments to induce the stem cell differentiation with a single or combination of grow factors [21]. Stem cells therefore were induced into one cell type within these environments. However, in vivo stem cells encounter a heterogeneous mixture of both chemoattractant and chemorepulsive signals [22]. A single administration of these bioactive factors may not be sufficient to yield the numbers of cells and cell types necessary to replace the ones lost due to organ failure, which often involves progressive multiple cell degeneration. Understanding the ectopic stem cell differentiation mechanisms is a key research interest due to its potential clinical applications [23]. A longterm bioactive factor delivery system, with appropriate release kinetics and tuning of relative stem cell differentiation, is essential for correct organ regeneration. Furthermore, among the various stem cell types Adipose-Derived Stem Cells (ADSCs) are easily harvested from patient themselves without immune rejections and have shown multidirectional differentiation potential without ethical controversy [24]. Studies have shown that human ADSCs have the ability to differentiate into osteogenic, adipogenic and neurogenic lineages [25].

Spatial effects of stem cell engagement in 3D printing constructs

In one of our previous studies, ADSCs were used to establish a multicellular system through a one-nozzle cell printing technique. Attempts were made to control the ADSCs differentiation into endothelial cells and adipocytes according to their positions within an orderly 3D construct. After the ADSCs were printed with a gelatin-based hydrogel (gelatin/alginate/fibrin), CaCl2 and thrombin solutions were used to crosslink/polymerize the alginate/fibrin molecules. The crosslinked/polymerized gelatin-based hydrogels have micro-porosity that is critical for fluid penetrating and bioactive factor exchanges during the in vitro engagement stages [26,27]. A Dulbecco’s modified Eagle’s medium (DMEM) culture medium containing 10% Fetal Bovine Serum (FBS), 1 mmol/L insulin, 10 ng/ mL Endothelial Growth Factor (EGF) and 50 U/mL aprotinin was used for 3 d to induce the ADSCs on the channels to differentiate into endothelial cells. Then the culture medium was changed to a DMEM containing 10% FBS, 1 mM insulin, 1 mM dexamethasone (DXM; Sigma), 0.5mmol/L isobutylmethylxanthine (IBMX; Sigma), and 50 u/mL aprotinin for 3 d to induce the ADSCs in the construct to differentiate into adipocytes. With these cocktail growth factor inducements, ADSCs in the construct were differentiated into endothelial cells and adipocytes respectively according to their positions in the construct. After the 3 days engagement with EGF, immunofluorescence staining affirmed that over 90% of the ADSCs on the walls of channels differentiated into mature endothelial cells (CD31 possitive) with typical tubular vessel structures. The mature endothelial cells connected each other to form vessel-like structures with endothelin-1/nitric oxide secretion abilities (Figure 1). After another 3 d engagement with insulin, DXM and IBMX, Oil red O staining confirmed that ADSCs in the construct differentiated into adipocytes with a spherical shape and adipokine (leptin) secretion ability (Figure 2a, 2c, 2e). The differentiations of the ADSCs were based on growth factor inducing (Figure 2c, 2d) and cell position in the 3D construct. Those ADSCs on the surface of walls were easily induced to differentiate into mature endothelial cells and form tubular structures throughout the engineered 3D constructs. While those ADSCs deep in the gelatin-based hydrogels were more sensitive to differentiate into adipocytes during the later inducement stage. In particular, the engineered adipose tissues secreted adipokine, such as leptin, a typical biomark of adipose tissue. The engineered endothelial cells released endothelin-1 (ET-1) and nitric oxide (NO), special biomarks of mature endothelial cells. These functions were coincident with those of in vivo intima and adipose tissues [26,27]. The advantages of this 3DP and engagement model are obvious. It is not only to provide a new approach to engineer orderly vascular adipose-tissues with a predesigned endothelial network, but also to establish a method to evaluate the interaction effects of heterotypic cell - bioactive factor, cell - cell and cell - matrix on different levels.