**4. Some outstanding achievements made in Tsinghua University**

In parallel with the above mentioned RP approaches, a series of RP technologies have been explored extensively by Professor XH Wang's group at the Center of Organ Manufacturing, Department of Mechanical Engineering, Tsinghua University, China. State-of-the-art of the layer-by-layer modeling, material incorporation, and manufacturing principles of these tech‐ niques can be found in some of the pertaining references. The advantages and disadvantag‐ es of these approaches to be used in complex organ manufacturing have also been listed in Table 2. Previous studies have demonstrated their abilities to engineer complex 3D tissues using various single/double nozzle/syringe RP systems. In the following section some tech‐ nical specifications are highlighted.

The Integrations of Biomaterials and Rapid Prototyping Techniques for Intelligent Manufacturing of Complex Organs http://dx.doi.org/10.5772/53114 447

446 Advances in Biomaterials Science and Biomedical Applications

**Figure 3.** Several unique intelligent rapid prototyping devices and their functional cell-laden products: A) The inkjet cell printer and its bagel-like quasi-3D structure developed in Clemson University, prof. T Boland's group [47]. B) The robotic printing platform and its crescent construct made in Cornell University, prof. LJ Bonassar's group [49]. C) The direct-write system and its preliminary 3D figures developed in University of Arizona, prof. SK Williams' group [50]. D) A modular tissue printing platform with 4 'cartridges' to load cell suspensions and hydrogel precursors developed in Brigham and Women's Hospital, Harvard Medical School, Prof. S.-S. Yoo's group [65]. E) A bioprinting tubular struc‐ ture with cellular cylinders developed in University of Missouri, Columbia, USA, Prof. G Forgacs' group [66]. F) A laserguided direct writing (LGDW) system and its patterned factor-linked beads on a stem cell monolayer with micrometer accuracy (Bar = 200 μm) developed in University of Minnesota, prof. D.J. Odde's group [67].

#### **4.1. The single syringe cell assembling technique**

Figure 4 shows some of the cell assembling results using our first generation cell assembling system. A gelatin based hydrogel system, such as gelatin, gelatin/chitosan, gelatin/hyaluron‐ an, gelatin/alginate, gelatin/fibrinogen or gelatin/alginate/fibrinogen, was integrated with a single syringe cell-assembling machine to obtain the necessary space and stabilizing factors for cell survival and tissue formation [53-58]. A single cell type was deposited at an ambient tem‐ perature (1~10℃) layer by layer in a chamber as the sol state material was transferred into a hy‐ drogel. Grid hepatic tissues, endodermis, and adipose tissues have been regenerated by using this single syringe cell-assembly machine at about 8℃. The gelatin based hydrogel network provided stabilization support for the 3D constructs during the fabrication and post culture stages. This mild deposition temperature is favorable for biological property preservation as increased Joule heating can result in loss of cell viabilities and bioactivities. During the culture period, the gelatin based hydrogel served as both a mass transportation template for tissue de‐ velopment and an extracellular matrix accommodation mimicking the microenvironment in native organs. The use of the natural gelatin based hydrogels was clearly highlighted the dis‐ tinct advantage of this cell assembly technique for fabricating living tissue analogs. A shortage of the single nozzle/syringe systems was that, these systems lack the ability to easily create parts with spatial heterogeneous materials. Consequently, two double nozzle/syringe RP sys‐ tems have been explored to deposit different materials at different temperatures.

**Figure 4.** Hepatocyte and adipose-derived stem cell (ADSC) assembling based on the first generation of cell assem‐ bling technique developed in Tsinghua University, prof. XH Wang' group [53-58]

#### **4.2. The double syringe cell assembling technique**

**4.1. The single syringe cell assembling technique**

448 Advances in Biomaterials Science and Biomedical Applications

Figure 4 shows some of the cell assembling results using our first generation cell assembling system. A gelatin based hydrogel system, such as gelatin, gelatin/chitosan, gelatin/hyaluron‐ an, gelatin/alginate, gelatin/fibrinogen or gelatin/alginate/fibrinogen, was integrated with a single syringe cell-assembling machine to obtain the necessary space and stabilizing factors for cell survival and tissue formation [53-58]. A single cell type was deposited at an ambient tem‐ perature (1~10℃) layer by layer in a chamber as the sol state material was transferred into a hy‐ drogel. Grid hepatic tissues, endodermis, and adipose tissues have been regenerated by using this single syringe cell-assembly machine at about 8℃. The gelatin based hydrogel network provided stabilization support for the 3D constructs during the fabrication and post culture stages. This mild deposition temperature is favorable for biological property preservation as increased Joule heating can result in loss of cell viabilities and bioactivities. During the culture period, the gelatin based hydrogel served as both a mass transportation template for tissue de‐ velopment and an extracellular matrix accommodation mimicking the microenvironment in native organs. The use of the natural gelatin based hydrogels was clearly highlighted the dis‐ tinct advantage of this cell assembly technique for fabricating living tissue analogs. A shortage of the single nozzle/syringe systems was that, these systems lack the ability to easily create parts with spatial heterogeneous materials. Consequently, two double nozzle/syringe RP sys‐

tems have been explored to deposit different materials at different temperatures.

**Figure 4.** Hepatocyte and adipose-derived stem cell (ADSC) assembling based on the first generation of cell assem‐

bling technique developed in Tsinghua University, prof. XH Wang' group [53-58]

Different from the above single syringe cell assembling technique, a double syringe cell assem‐ bling technique was developed in Tsinghua Unversity with a updated software and hard‐ ware. Gradient and cylindrical architectures consist of two different cell-laden hydrogels have been fabricated at a temperature range of 8 – 10℃ [59,60]. Two cell lines encapsulated in the similar gelatin-based hydrogels were put into different regions or compartment in a construct (Figure 5). The embedded branched networks enable culture medium to flow through the en‐ tire construct with unparalleled geometric complexity. However, there is a fatal shortcoming of this system to be used in complex organ manufacturing. The mechanical weak properties of the gelatin-based hydrogel made it impossible to connect the branched construct to an *in vivo* vascular system to endure anti-suture anastomosis and blood pressure even after a long-term *in vitro* culture period.

**Figure 5.** Cell assembling based on a two syringe RP technique developed in Tsinghua University, prof. XH Wang' group. Two different cell types in the gelatin-based hydrogels can be assembled simultaneously into a construct [59-60].

#### **4.3. The combination of cell assembly and cryopreservation techniques**

With the advantages of the gelatin-based hydrogel, cryoprotectants (e.g. dimethyl sulfoxide (DMSO), glycerol, and dextran-40) can be incorporated into the cell/hydrogel system and the constructs can be stored at low temperature (below -80℃) directly after the fabrication stage (Figure 6). This incorporation technique represents a significant advancement towards the cell-laden product storage and transport, potentially resulting in labor and resource saving, clinical availability and medical convenience [80-82]. With the gelatin-based hydrogel vari‐ ous bio-factors including macromolecular cell growth factors, small chemical regulators, and even genes/drugs can be easily incorporated to the deposition or assembling systems. This approach is suitable for some special natural thermosetting polymers' (e.g. gelatin and agar‐ ose) deposition and opens a new avenue for complex organ manufacturing.

**Figure 6.** The combination of cell assembly and cryopreservation techniques, developed in prof. XH Wang' group [80-82].

#### **4.4. The double-nozzle low-temperature deposition manufacturing (DLDM) system**

The creation of a geometrically complex branched vascular system is a subject of broad fun‐ damental and technological interest in complex organ manufacturing. With the DLDM sys‐ tem it is easy to deposit two different material systems, especially both synthetic and natural polymer systems simultaneously in a construct (Figure 7). Grid, tubular and elliptic struc‐ tures with both synthetic and natural polymers, such as PU/gelatin and PU/collagen, have been produced at a low-temperature range of -20 - -30 ℃ [61-64]*.* As shown in Figure 7C, PU constructs can be stored at low temperature (below -80℃) directly after the fabrication stage (Figure 6). This incorporation technique represents a significant advancement towards the cell-laden product storage and transport, potentially resulting in labor and resource saving, clinical availability and medical convenience [80-82]. With the gelatin-based hydrogel vari‐ ous bio-factors including macromolecular cell growth factors, small chemical regulators, and even genes/drugs can be easily incorporated to the deposition or assembling systems. This approach is suitable for some special natural thermosetting polymers' (e.g. gelatin and agar‐

**Figure 6.** The combination of cell assembly and cryopreservation techniques, developed in prof. XH Wang' group

The creation of a geometrically complex branched vascular system is a subject of broad fun‐ damental and technological interest in complex organ manufacturing. With the DLDM sys‐ tem it is easy to deposit two different material systems, especially both synthetic and natural polymer systems simultaneously in a construct (Figure 7). Grid, tubular and elliptic struc‐ tures with both synthetic and natural polymers, such as PU/gelatin and PU/collagen, have been produced at a low-temperature range of -20 - -30 ℃ [61-64]*.* As shown in Figure 7C, PU

**4.4. The double-nozzle low-temperature deposition manufacturing (DLDM) system**

[80-82].

ose) deposition and opens a new avenue for complex organ manufacturing.

450 Advances in Biomaterials Science and Biomedical Applications

and collagen were successfully assembled into a tubular double layer construct. In Figure 7D, an elliptic hybrid hierarchical PU-cell/hydrogel construct with branched and grid inter‐ nal channels was realized. Cells can survival the heterogeneous fabrication, polymerization/ crosslinking, and even storage stages with a high recovery proliferation ability. Figure 7D demonstrates that the external out coat was made of a PU/tetraglycol solution to provide mechanical support for the whole construct. The internal branched and grid channels were made of a cell/dimethyl sulfoxide (DMSO) containing gelatin/alginate/fibrinogen hydrogel to encapsulate ADSCs. Both the out coat PU and compartment cell/hydrogel layers possess microporous, which permit water, oxygen and other small molecules to pass. During the fabrication stage, a low temperature in the range of -20 - -30 ℃ around the nozzles is an im‐ portant factor to control the sol-gel transformation of the material systems. If the tempera‐ ture is set too high, the deposited fiber (strand) cannot solidify to form a stable 3D structure. On the other hand, if the temperature is set too low, the fiber is frozen too quickly to fuse with the previous deposited layer. An optimum deposition temperature has played a central role in putting the heterogeneous material systems at the desired locations in the construct.

**Figure 7.** A DLDM technique developed in Tsinghua University, prof. XH Wang' group. An elliptical hybrid hierarchical polyurethane and cell/hydrogel construct was fabricated using the DLDM system [61-64].

This DLDM technique has demonstrated some outstanding merits in complex organ manu‐ facturing with two different material systems that are technologically and biologically inca‐ pable to produce using the other existing or traditional RP techniques. The potential applications of the assembled elliptic hybrid hierarchical constructs are diverse, such as cellcell interaction analyses, stem cell differentiation tracing (pursuing), chemical drug screen‐ ing, and pathogenic mechanism studies. The synthetic PU system can provide elaborate compartments for cell/hydrogel accommodation. In these compartments, the composition of the cell/hydrogel mixture becomes the key factor in ensuring spatially uniform cell distribu‐ tion, survival, proliferation and differentiation. By encapsulating the cell/hydrogel mixture in the PU compartments, the composition and proportion of hydrogel components can be easily adjusted to meet the necessary requirements for mimicking the natural cellular ar‐ rangements. A maximal cell density (hydrogel-poor and cell-rich) can be easily achieved in the compartment. The use of gelatin-based hydrogel can even be avoided completely in this system, irrespective of stabilization of the construct. Compared with the pure cell/gelatin/ alginate/fibrin construct made by the single/double RP systems, the hybrid hierarchical net‐ work can provide much higher mechanical stability and pressure resistance abilities when it is applied to *in vitro* pulsatile cultures and *in vivo* blood vessel anastomoses. Some experi‐ ments have proved that the 3D constructs with intrinsic interconnected branched and grid channels were easily adapted to an *in vitro* pulsatile culture and *in vivo* implantation system [83-86].
