**5. A four-nozzle low-temperature deposition manufacturing (FLDM) system**

At present, a FLDM system is under development in professor XH Wang's group [18]. Fig‐ ure 8 demonstrates the outlook of the machine and a primary try on a liver lobe like struc‐ ture construction. Compared with the DLDM RP system, two more nozzles have been equipped. Thus, two more cell types can be incorporated simultaneously into a construct. This amplified integration possesses some outstanding advantages towards complex organ manufacturing: (i) hierarchically organization of multiple population of cells and growth factors in a more intricate physiologically mimicking geometry; (ii) simultaneously deposi‐ tion of one scaffold material, a vascular system with two main cell types, and one parenchy‐ mal cell type in a more elegant native tissue-specific phenotype; (iii) computer definition of the fluid paths and macro/microstructures in a more patient specific manner; and (iv) spatial distribution of multi-tissue boundaries and fluorescent biomarkers in a more controllable pattern. This FLDM RP system makes it possible to partially control over the design, model‐ ing and fabrication of a highly hierarchical liver lobe like construct in a rapid, convenient, and cost effective manner.

The Integrations of Biomaterials and Rapid Prototyping Techniques for Intelligent Manufacturing of Complex Organs http://dx.doi.org/10.5772/53114 453

**Figure 8.** A schematic description of the modeling and manufacturing processes of four liver lobe-like constructs with a FLDM RP system developed in Tsinghua University, prof. XH Wang' group [18].

### **6. Emphases to some technical specifications**

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

452 Advances in Biomaterials Science and Biomedical Applications

**5. A four-nozzle low-temperature deposition manufacturing (FLDM)**

At present, a FLDM system is under development in professor XH Wang's group [18]. Fig‐ ure 8 demonstrates the outlook of the machine and a primary try on a liver lobe like struc‐ ture construction. Compared with the DLDM RP system, two more nozzles have been equipped. Thus, two more cell types can be incorporated simultaneously into a construct. This amplified integration possesses some outstanding advantages towards complex organ manufacturing: (i) hierarchically organization of multiple population of cells and growth factors in a more intricate physiologically mimicking geometry; (ii) simultaneously deposi‐ tion of one scaffold material, a vascular system with two main cell types, and one parenchy‐ mal cell type in a more elegant native tissue-specific phenotype; (iii) computer definition of the fluid paths and macro/microstructures in a more patient specific manner; and (iv) spatial distribution of multi-tissue boundaries and fluorescent biomarkers in a more controllable pattern. This FLDM RP system makes it possible to partially control over the design, model‐ ing and fabrication of a highly hierarchical liver lobe like construct in a rapid, convenient,

[83-86].

**system**

and cost effective manner.

Theoretically, RP technology is able to produce any required complex shape. The standard modeling and deposition technologies enable the hybrid hierarchically ordered patterns to be generated in an automatic, convenient, and inexpensive manner. Again, we use the liver as an example. In a liver lobe at least 6 different cell types are structured as repeated units. These units can achieve high oxygen exchange and nutrient supply for a mass of cells where the cell sizes are in the range of ~20 μm. This geometry enables a high degree of processing optimization, which provides the opportunity for RP designers and manufacturers to con‐ trol readily the distribution of different cells in a construct. Stimulated by this motivation, many groups have tried different RP systems with only thin or quasi-3D cell containing structures so far*.* Someone even claims to use scaffold free cell aggregates to print organs. This has been proven to be a time-consuming process and cells can not find their respective places in a complex organ without the support of scaffold materials.

Different from all the existing techniques, the gelatine-based hydrogel has been explored ex‐ tensively as an internal scaffold material with the single/double syringe/nozzle RP techni‐ ques in the author's own group Tsinghua University. Aqueous gelatin solution is an amorphous natural hydrogel in which cells can be encapsulated, extruded and deposited at desired positions [87-90]. This solution is flexible with a gelation temperature of 20℃ and allow the diffusion of hydrophilic substrates. The sol-gel transform property makes it possi‐ ble to deposit the gelatin-based cell-laden hydrogel at a large range of temperature (from 20℃ to -30℃). However, this hydrogel is not stable at 37℃. The mechanical properties of the gelatin-based hydrogels are notoriously inadequate and prohibit the use in stress-loaded im‐ plants. To improve the stability of the gelatin-based hydrogels, alginate and fibrinogen were incorporated. Sodium alginate (composed of mannuronic and guluronic (G) dimmers) is a biocompatible and biodegradable polymer, and has been widely used in cell encapsulation technology, although the biocompatibility of the alginates in relation to their composition is a matter of debate. Sodium alginate can be reversibly cross-linked by divalent cations, such as Ca+2 and Mg+2, to form a relatively stable hydrogel. Fibrin, derived from blood fibrinogen, is another natural biocompatible and biodegradable polymer, which has been widely used as sealant and adhesive during surgery. With the catalyzing of thrombin smaller fibrinogen molecules can polymerize to form a stable fibrin hydrogel.

In addition to being able to build complex structures with precision and accuracy, it is equally important that the mechanical properties of the supporting materials are suitable for the intended applications. A novel linear elastomeric polyurethane from soft polycaprolac‐ tone (PCL) and polyethylene glycol (PEG) segments, and hexamethylene diisocyanate (HDI) chain extender has been synthesized in the authors' own group and used as an external scaf‐ fold material. This PU possesses tunable biodegradability, excellent biocompatibility and compatible mechanical properties with animal veins [91,92]. Long-term *in vivo* biocompati‐ bility and biodegradability of the PU have been proven with a rabbit model. It has success‐ fully repaired nerve and vein defects without any detected side effects, such as thrombosis, inflammation, intimal hyperplasia, and calcification. The excellent mechanical properties, bi‐ ocompatibilities, adjust abilities and processing abilities have made this kind of polymer to be outstanding from the other existing synthetic scaffold biomaterials, such as polyhydroxy‐ butyrate (PHB) [93], poly(D,L-lactic-co-glycolic acid) (PLGA) [94], and poly(tetrafluoroethy‐ lene) (PTFE) [95].

To date, the most widely recognized advantage of the RP technology (i.e. layered manufac‐ turing methodology) is the relative ease of automatically manufacture of complex geometric shapes with heterogeneous structures composed of multi-material regions. Complex organ manufacturing aims to automatically produce complex organs directly from CAD models with high sophisticated RP techniques. Since the earlier concept of complex organ manufac‐ turing using both synthetic and natural scaffold biomaterials and multi-nozzle RP techni‐ ques was first introduced in 2007*,* the present technique was developed gradually [13-18]*.* As displayed in Figure 7D, the virtual elliptic construct with branched fluidic network has been designed and fabricated according to a pre-defined CAD software. The integration of the DLDM RP technique with the cell cryopreservation technique together with the mechan‐ ically strong enough synthetic PU scaffold enables us to efficiently produce spatially hetero‐ geneous cell-laden tissue/organ substitutes that would otherwise be challenging to achieve [61-64]. This integrated technique therefore has the potential to lead a big revolution in the fields of tissue engineering and regenerative medicine.

It is expected that in the following several years these integrated RP technologies will see their major break-through development stage and play a key role in complex organ manu‐ facturing area. With the proper integrations of biomaterials and enabling RP techniques, it is possible for us to address all the challenges involved in complex organ manufacturing and to make the realization of complex organ manufacturing both feasible and practical. These proper integrations also benefit some of other related areas, such as high throughput drug screening, stem cell differentiation induction, fluorescent dye discovering, energy metabolite model establishing and cancer/stem cell behavior controlling.
