**3. The integrations of biomaterials with RP techniques for complex organ manufacturing**

The ability to put material only into a specific location where it is desired could have a pro‐ found impact on how parts are designed and manufactured [46]. Similarly, the ability to put different biomaterials (including different cell types) to exact sites where they are desired could have a profound impact on how complex organs are designed and manufactured. For example, in a complex organ, such as the liver, at least three different cell types (hepato‐ cytes, stellate cells, and Kupffer cells) are required that function in a construct along with the three common cell types of a vascular system. The fundamental unit of the liver, the acinus, has a typical radius of 500 μm. Within this structure at least six cell types interact with one another to coordinate the diversity of liver functions [46]. The spatially heterogeneous ar‐ rangements of multi-tissues make all the traditional, or existing techniques incapable of completing this ambitious task.

Over the last ten years, the integration of biomaterials with RP techniques in creating special 3D constructs for various biomedical applications has emerged. The ability to use data from clinical imaging techniques like magnetic resonance imaging (MRI), computerized tomogra‐ phy (CT) or patient-specific data makes RP techniques particularly useful for biomedical ap‐ plications. Several research groups have adapted different RP techniques to assemble (or print) cell-laden constructs directly from computer-programmed design models with high resolution (Table 2) [47-64]. Six unique intelligent RP devices as well as their primary prod‐ ucts are shown in Figure 3[47-52, 65-67]. These processes have demonstrated some possibili‐ ties in the area of complex organ manufacturing. The pros and cons of these techniques in complex organ manufacturing are outlined in Table 2. Those with only porous 3D scaffolds are not included here because these integrations have been reported extensively in the for‐ mer reviews [36,37,44,68-79].

**Figure 2.** A "family tree" indicates various integrations of biomaterials and rapid prototyping techniques.

442 Advances in Biomaterials Science and Biomedical Applications


**Table 2.** Comparison of different cell-laden rapid prototyping techniques in complex organ manufacturing

Due to the heterogeneous properties of complex organs both in geometrical structures and material components, emphases should be given to those RP techniques with further devel‐ opment possibilities in the further integrations of biomaterials and equipments. In the fol‐ lowing part of this section, some special two or multiple syringe/nozzle techniques are highlighted. In Harvard Medical School, Lee and coworkers have printed a collagen hydro‐ gel precursor, fibroblasts and keratinocytes into a quasi 3D structure for skin repair using a robotic platform (Figure 3D) [65]. The procedure involves printing a layer of liquid collagen to act as a hydrogel precursor. The liquid collagen is crosslinked with a nebulized aqueous crosslinking agent (sodium bicarbonate) to form a hydrogel that provides structural integri‐ ty for the subsequent cell suspensions. In fact, this technique is an extension of the above mentioned 3DP or 3DB robotic system with additional syringes as "cartridges" to load two cell suspensions and hydrogel precursors. Highly viable proliferation of each cell layer (85% for keratinocytes and 95% for fibroblasts) was observed on both planar and non-planar sur‐ faces. For thin tissue/organ (such as skin/bladder) manufacturing, this technique is a right choice. However, for complex organ manufacturing, some intrinsic shortcomings, such as limited printing height, and difficult to control the collagen gelation process, made this tech‐ nique almost incapable.

**Technique Pros Cons Refs**

Complex 3D constructs are difficult to realize; limited feature height (< 5 µm); lack of structural support for cell layer or cell aggregates; tissue formation into lines depends on cell or cell aggregate fusion or assembling; poor mechanical properties.

Cell viability depends largely on the inner diameter of the gauge tip, collagen concentration and extraction environments; difficult to control the

Limited materials can be used; limited height of 3D construct (< 10 µm); difficult cell-cell interactions; poor mechanical

Limited materials can be used; weaker

Material viscosity and temperature

mechanical properties.

collage gel state.

properties.

dependent.

[47, 48]

[49, 50]

[51, 52]

[53 - 60]

[61 - 64]

Several thermosensitive hydrogels can be used as biopaper; low viscosity cell suspensions or aggregates can be used as bioink; Cell viability greater than 85%.

444 Advances in Biomaterials Science and Biomedical Applications

Low and high viscosity hydrogels, including type 1 collagen and alginate can be used; high cell viability (up to 98%); flexible

High viscosity hydrogels, such as Pluronic F127, Matrigel, alginate and agarose, can be used; multiple cell types can be incorporated; homogeneous and heterogeneous structures can be created.

Gelatin-based hydrogels can be used; a wide range of biological components can be incorporated; variable and hybrid geometric shapes; high cell viability (more than 98%); easy for long-term storage

and transportation.

A wide range of biomaterials including both synthetic and natural polymers can be used; a wide range of biological components can be incorporated; arbitrarily hybrid geometric shapes; high mechanical properties; easy for long-term storage and transportation.

**Table 2.** Comparison of different cell-laden rapid prototyping techniques in complex organ manufacturing

Due to the heterogeneous properties of complex organs both in geometrical structures and material components, emphases should be given to those RP techniques with further devel‐ opment possibilities in the further integrations of biomaterials and equipments. In the fol‐ lowing part of this section, some special two or multiple syringe/nozzle techniques are highlighted. In Harvard Medical School, Lee and coworkers have printed a collagen hydro‐ gel precursor, fibroblasts and keratinocytes into a quasi 3D structure for skin repair using a

geometric shapes.

3D inkjet bioprinting (3DP) in and Pittsburgh Clemson University, USA

3D direct-write bioprinting in University of Cornell and Arizona, USA.

3D fiber deposition (3DF) in University Medical Center Utrecht, The Netherlands.

3D single/double syringe cell assembling (or pressure assisted manufacturing (PAM)) in Tsinghua University, China.

Double-nozzle lowtemperature deposition manufacturing (DLDM) in Tsinghua University, China.

In University Medical Center Utrecht, The Netherlands, Prof. J Alblas's group, a special bio‐ scaffolder pneumatic dispensing system (SYS + ENG) was adapted for printing cell-laden bone tissue repair hydrogels. High viscosity alginate (10% w/v) and BD MatrigelTM (10% w/v) hydrogels were employed. A limited ten-layer rectangular 3D construct of 10 ×10 mm with spacing between fibers of 0.8-2.5 mm and a thickness of 100 μm was fabricated and subsequently crosslinked in a CaCI2 solution [51]. In spite of the limited height, the intercon‐ nected channels are still necessary for oxygen and nutrient delivery, as well as for tissue for‐ mation and vascular ingrowth. There are two critical drawbacks of this technique in complex organ manufacturing. The first is the poor mechanical properties of the cell-laden alginate or matrigel hydrogel for use as vascular systems. The second is that low viscosity hydrogels (including alginate and matrigel) are hard to be assembled into 3D constructs.

In university of Missouri, Norotte and coworkers used agarose rods as a molding template to print multicellular spheroids with their special bioprinter to form a tubular cell-laden structure (Figure 3E) [66]. After the fabrication stage, they manually pulled the agarose rods out of the tube, and concluded that it is a time consuming and labor-/spheroid- intensive procedure.
