**7. Concluding remarks**

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

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‐

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‐

molecules can polymerize to form a stable fibrin hydrogel.

454 Advances in Biomaterials Science and Biomedical Applications

lene) (PTFE) [95].

The goal of complex organ manufacturing is to directly fabricate multiple cell types into an organ substitute using a multiple nozzle RP system. Analogous to the process of building a nuclear power plant, complex organ manufacturing requires the ability to control the posi‐ tions of many cell types, internal/external scaffold materials, and even cell growth factors on the nano-, micro-, and macro-scales with respect to each others. The integrations of biomate‐ rials and RP techniques have significantly improved the ability to manufacture cell-laden constructions with predefined geometries under the instructions of CAD models or medical data (for example, patient-specific images). Especially, recent advances in DLDM and FLDM RP techniques in Tsinghua University have leveraged these progresses. Although still at its infant stages and associated with numerous problems, ever-increasing evidence supports the intriguing hypothesis that the integrations of multiple biomaterials (including multiple cell types) and multiple nozzle RP techniques will eventually change the traditional practi‐ ces and make the dreams of complex organ manufacturing come true. It is expected that in the future, most of the reconstructive disciplines of complex organ manufacturing will be fully revised by the development of new multiple nozzle RP systems with optimal safety, easy manipulation ability and maximum reliability. Multiple nozzle RP techniques will un‐ doubtedly play an important role in the future complex organ manufacturing area. Cells in the engineered construct will potentially behave as comfortably as in their natural *in vivo* environment. Further studies are therefore needed to elucidate and determine the funda‐ mental structure-function relationships of diverse tissues in a complex organ, the nutrition supply systems and the heterogeneous structural cues to promote full functional realization in a complex organ. Ever increasing evidences have indicated that with the right integra‐ tions of biomaterials and RP techniques, a brand-new era of complex organ manufacturing like the rising sun, is on the horizon.
