**4. Bioprinted tissues**

#### **4.1 Neural**

Injuries of the central and peripheral nervous system can be challenging to repair. The central nervous system does not regenerated under normal conditions, and any injuries it sustains can lead on to neurogenerative disease. Damage to the peripheral nervous system is mostly treated with autografts and allografts, however motor and sensory recovery rates are poor. In a review of studies recording sensory recovery after digital nerve repair, only 67% of patients reported good sensory repair or better with a nerve graft, which increased to 79% of patients with artificial conduits [33]. This result indicates the potential for synthetically made nerve guidance conduits and therefore the use of bioprinting.

Some of the initial work involving bioprinting of mammalian cells was performed using Chinese hamster ovary (CHO) and rat embryonic motoneuron cells [34]. It was established that using an inkjet printer to print the cells did not affect cell proliferation or the polarized morphology of the motoneuron cells. However,

 evaluation of cell lysis immediately after printing indicated that ~8% of the cells were damaged due to the printing process. Further work with rat primary embryonic hippocampal and cortical neurons demonstrated a cell viability of 74.2 ± 6.3%, as well as the maintenance of basic function, phenotype and electrophysiological properties [35].

Lorber et al. inkjet printed cells from the mature adult nervous system [36]. Monitoring the cells during droplet ejection, the cells did not undergo major distortion and hence destruction caused by shear forces. It was shown that printed glia cells retained their ability to support the growth of the seeded retinal ganglion cells, which is an essential interaction for maintaining proper nerve function. Tse et al. printed a combination of neuronal and glial cells [37]. High cell viabilities >86% for neuronal cells and >89% for Schwann cells were achieved. Not only did the printed cells exhibit early neurite growth in comparison to non-printed cells, neurites also grew longer in length.

Using an extrusion printing process, neural stem cells (NSCs) have been printed embedded in polyurethane (PU) [38]. The bioprinted scaffolds restored nerve function when used to treat Zebra fish with a traumatic brain injury. Owens et al. developed the extrusion bioprinting process to produce purely cellular nerve grafts [39, 40]. Mouse bone marrow stem cells (BMSCs) and Schwann cells (SC) were extruded alongside agarose rods. The agarose rods were removed after a 7 day maturation period leaving a tubular cell structure. Implanted to repair a sciatic nerve injury in rats, both motor and sensory function was restored after 40 weeks.

SLA has been used to print human neural stem cells (hNSCs) [41]. Scaffolds were made containing GelMA and graphene nanoplatelets. GelMA content had a negative effect on cell proliferation rates, while the presence of graphene, known to stimulate hNSCs into neural differentiation [42], improved the cellular response. By monitoring glial fibrillary acidic protein (GFAP) expression, which is associated with the development of ependymal cells, and ß-tubulin III, which is associated with neurons, it was determined that the printed cells had differentiated over a 14 day period post-printing.

#### **4.2 Skin**

In the repair of deep partial thickness and full thickness burns a new epidermal barrier needs to be established, this is usually achieved through transplantation [43]. Current treatment techniques, including split thickness grafts, lack the inclusion of structures such as hair follicles, subcutaneous glands or sweat glands and do not provide the full restoration of sensory and motor neurons [43]. This can cause problems for the patient, especially the lack of sweat glands, which can prevent proper thermo-regulation. Bioprinting can be used to develop a complex tissue as multiple inks can be printed at once. Although the production of replica human skin has yet to be achieved, there have been many promising advances for the treatment of full thickness wounds.

 Laser-assisted bioprinting was tested for the printing of different skin cells: fibroblasts and keratinocytes, as well as human mesenchymal stem cells (hMSCs) [25]. Printing was shown not to induce apoptotic activity and cell viabilities were measured as >90%. The hMSCs maintained their phenotype, implying that the transfer process did not induce differentiation of the cells.

Kim et al. bioprinted a skin model using both extrusion and inkjet technologies [44]. A collagen/PCL ink was extruded to form the basis of the scaffold, followed by the inkjet printing of primary human dermal fibroblasts (hFBs) and primary human epidermal keratinocytes (hKCs). Using inkjet printing to deposit the cells provided a controllable and even distribution. When placed in cell culture over a

#### *Bioprinting DOI: http://dx.doi.org/10.5772/intechopen.81135*

14 day period, the bioprinted construct developed biological characteristics such as elongated hFB, and dermal and epidermal layers.

 A PEG based skin model was produced by Rimann et al. [45] using a microvalve droplet system. Alternate layers were printed of a PEG-based ink followed by the hFBs ink suspension. Each layer of the PEG based ink was immediately polymerized using UV light. Due to the use of UV light the cells were analyzed for UV damage. By comparing printed and unprinted cells, it was shown that the cell DNA was not damaged during the printing process. The cells adhered to the structure, maintaining a typical morphology and were well dispersed within the polymer matrix. After a 3-week period, the cells continued to proliferate and produce their own extra cellular matrix, however, subsequent seeding of hKCs on top of the printed scaffolds did not achieve a fully stratified epidermis when exposed to the air-liquid interface for 14 days.

 A more complex bioprinted skin model has been achieved by printing multiple cell types [16, 46, 47]. Using a microvalve droplet system, initial investigatory work used both hFBs and hKCs suspended in cell media that were printed in-between layers of a printed collagen gel [46, 47]. Using a droplet based technique enabled the precise placement of cells, with a recorded 68 ± 13 hKCs per droplet and 93 ± 13 hFBs per droplet for each respective ink suspension. Compared to skin tissue scaffolds that had used a manual seeding technique, the bioprinted scaffolds retained their shape better and developed denser epidermal layers. The epidermal layers formed tight junctions between neighboring cells, an important feature of the epidermis for providing an effective barrier.

Further work included pigmentation of the printed skin model by printing human epidermal melanocytes (hMCs) into the epidermal layer [16], demonstrated in **Figure 3**. The hKCs reached a terminal differentiation to form a stratum corneum-like structure. hMCs started producing melanin which accumulated at hKC interfaces, causing a light pigmentation. When the hMCs were printed as a uniform layer, they formed nevus-like structures (freckles), however it was suggested that this could be prevented by controlling hMC cell densities to create a more even spatial distribution.

A collagen skin scaffold containing hFCs and printed human dermal microvascular endothelial cells (hMVECs), that was subsequently seeded with neonatal hKCs mixed in collagen, was compared to a commercial graft (Apligraf®) for treating full thickness skin wounds in mice [48]. After 14 days, the bioprinted scaffolds had adhered to the wound while the Apligraf® had dried out and detached from the implant site, although no infections were recorded for any of the test groups. Complete closure of the wound occurred first for the bioprinted scaffold after 21 days, followed by the Apligraf® after 28 days. Wound sites treated with either the commercial graft or the bioprinted graft developed epidermal and dermal

#### **Figure 3.**

*Bioprinted pigmented skin model fabricated using a microvalve droplet technique [16]. Fibroblasts are used for a dermal layer, and keratinocytes and melanocytes for an epidermal layer.* 

layers, however the bioprinted skin graft was shown to have the most histologically similar appearance to native skin, and developed a micro-vessel network.

Skardal et al. successfully demonstrated *in-situ* bioprinting to repair full thickness skin wounds on the backs of mice [49]. Amniotic fluid-derived stem (AFS) cells and bone marrow-derived mesenchymal stem cells (MSCs) were separately suspended in fibrinogen-collagen solutions and printed into the wound sites. The fibrinogen-collagen solution was crosslinked by printing a thrombin solution between each layer. Printing the skin graft directly into the wound site achieved complete coverage of the wound and a tight seal between the skin and graft. At all points during the study, wound contraction and re-epithelialization were significantly higher in wound sites treated with bioprinted cells. Printed scaffolds exhibited re-epithelialization of up to 89% which had organized, well defined layers, while the control only exhibited a 51% re-epithelialization with a poor structural quality.

#### **4.3 Cardiovascular**

Cardiovascular disease is one of the major causes of death globally. According to World Health Organization (WHO), cardiovascular disease accounts for more than one third of the total deaths each year [50, 51]. Depending on severity, treatment solutions may require replacement of damaged vital vasculature, or even heart transplantation. Problems such as organ shortages or requirements for multiple invasive surgeries mean that alternative solutions need to be found.

Different methods have been explored for the bioprinting of a 3D vascular network. Endothelial cells have been successfully printed with a high degree of accuracy using an inkjet printer. It was shown that during printing, a precise number of cells were ejected per drop [52]. Boland et al. used inkjet printing to print a crosslinking agent (calcium chloride) into an alginate/gelatin solution to produce vascular structures [53]. By printing a crosslinking agent into the polymer solution, a structure was formed out of hollow shells providing a microscopic porosity that proved beneficial for cell migration.

Cui et al. used inkjet printing to encapsulate human microvascular endothelial cells (hMVEC) in a fibrin hydrogel [54]. Scaffolds were fabricated with micronsized fibrin channels that had similar mechanical properties to other tissue engineered blood vessels. In cell culture, the cells aligned along the channels in a confluent lining, and had formed a ring-shaped microvasculature that sealed the fibroin channel with a high level of integrity.

Extrusion bioprinting has been used to print heart valve conduits [55]. Scaffolds were fabricated using a bioink of methacrylayed hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel) with suspended human aortic valvular interstitial cells (hAVIC). In cell culture, cells adhered and formed a monolayer on the surface of the bioprinted structure while the encapsulated cells below the surface started to remodel the hydrogel after 3 days.

Initial work into printing cardiac tissue was performed using alginate hydrogel and primary feline adult and human H1 cardiomyocytes [56]. Inkjet printed in a half heart shape, scaffolds had a 1 cm inner diameter and two connected ventricles. Due to the printed porosity, the large structure enabled the transportation of nutrients, and meant that the cells remained viable when placed in cell culture. Using electrical stimulation, the cardiac cells were observed to contract rhythmically which in turn caused the whole structure to beat periodically.

The generation and use of human pluripotent stem cell (iPSCs) is of growing interest for many applications as they can differentiate into multiple cell lineages [50]. Zhang et al. demonstrated a bioprinting methodology to fabricate

 endothelialized myocardium using iPSCs [57]. GelMA and iPSC scaffolds were extruded and seeded with cardiomyocytes to induce the formation of myocardium. The fabricated scaffolds were capable of spontaneous and synchronous contraction using low level electrical stimuli.
