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Polyelectrolyte gelatin-chitosan hydrogel optimized for 3D bioprinting in skin tissue engineering

tion of complex 3D multicellular tissue constructs. can be prepared by the N-deacetylation of insoluble
Despite being in its stage of infancy, bioprinting has chitin in the presence of alkaline solution [21] . In the
already demonstrated great potential for fabrication of presence of lysozymes, chitosan undergoes in vivo
multi-layered skin [5–7] , cartilage [8,9] and liver con- degradation via enzymatic hydrolysis to form by-pro-
structs [10] . It was highly anticipated that production of duct, glucosamine, which does not pose any toxi-
less-sophisticated human tissues/organs such as skin city [22] . Furthermore, chitosan triggers hemostasis and
[3]
would be a reality in the near future . Some current accelerates tissue regeneration due to the migration of
works on bioprinting of skin constructs include fabri- inflammatory cells and activation of fibroblasts that
cation of hydrogel constructs consisting of different produce multiple cytokines [23] . Notably, chitosan-based
skin cells (keratinocytes and fibroblasts) [5,6] and in-situ biomaterials have antimicrobial properties which can
printing of skin cells and biomaterials directly over help to reduce the incidence of sepsis [24] . Chitosan
the wound site [11] . Contrary to the common miscon- powders are generally soluble at acidic pH and the
ception that skin is a relatively simple 2D tissue, the amine groups in chitosan are protonated at pH lower
thin layer of human skin has a unique pattern created than 6 to confer the poly-cationic behavior to chitosan.
by the natural compartmentalization of different types With increasing pH, the amine groups become depro-
of skin cells that are positioned relative to each other tonated to form insoluble chitosan polymer. This so-
at high degree of specificity [12] . This specific ar- luble-insoluble transition occurs at its pK a value aro-
rangement of skin cells is essential for cell-cell inte- und pH 6–6.5, which is dependent on degree of
ractions that initiate autocrine and paracrine signaling N-deacetylation and molecular weight [25] . Despite its
within the native human skin [13] . attractive properties, chitosan alone has poor printabil-
As skin cells (fibroblasts) are capable of producing ity [26,27] and further modifications are required to in-
their own ECM proteins, the bio-inks serve as tempo- crease the printability of chitosan-based hydrogels.
rary 3D templates to guide the tissue morphogenesis. Gelatin, which is commonly used for biomedical
Collagen type I, the most abundant ECM protein in applications, exhibits negative charges when the pH of
human skin, is widely used for bioprinting of skin medium is above its isoelectric point (pH iso = 4.7) [28] .
constructs. Most of the biomaterials used in those stu- As such, interactions between the positively charged
dies [5,6,14–16] were mainly collagen-based, which has ammonium ions from chitosan react with carboxylate
relatively poor printability. Lee et al. printed layers of groups from the ampholytic gelatin result in the for-
collagen to create a 3D bioprinted collagen construct mation of a polyelectrolyte complex. Prior works on
[5]
with stacking height of 1.2 mm . Another work polyelectrolyte gelatin-chitosan scaffolds/films [29–32]
demonstrated printing of multi-layered cell-laden col- have demonstrated great potential for skin tissue en-
lagen constructs on non-planar surface using nebulized gineering applications. The polyelectrolyte gelatin-
crosslinking reagent [15] . Only planar sandwich con- chitosan hydrogel did not experience significant con-
structs were fabricated using the valve-based tech- traction in the in-vitro cell culture test over 4 weeks [32]
nique due to the slow pH-dependent crosslinking of and also demonstrated potential antimicrobial activ-
collagen prior to printing of subsequent layers. Koch ity [33] . An in-vivo study over a period of 16 weeks re-
et al. printed layers of encapsulated keratinocytes and vealed that the chitosan/gelatin hydrogel was efficient
fibroblasts onto a decellularized dermal matrix sheet in inducing fibrin formation and vascularization at the
[6]
via laser-based method . The printed construct com- implant-host interface [34] . The polyelectrolyte gelatin-
prised high number of keratinocytes and fibroblasts chitosan scaffolds are commonly prepared via freeze-
(different from representative cellular density within drying [29,31,32] or solvent-casting approaches [29,30] .
native human skin) and there is no variation in the In this paper, gelatin was modified with chitosan to
extracellular matrix density across the depth of printed form polyelectrolyte gelatin-chitosan (PGC) hydrogels
structure [17] . to demonstrate its potential for bioprinting applica-
Progress in bioprinting of skin is severely hindered tions. The interactions between the chitosan and gela-
due to limited choices of printable biomaterials. Over tin within the polyelectrolyte complex were evaluated,
the recent years, the attractive traits of chitosan poly- followed by rheological characterization of the PGC
mer have gained huge attention for wound healing hydrogels at varying shear rates and temperatures.
applications [18–20] . Chitosan is a linear polysaccharide Next, different combinations of printing pressures and
of D-glucosamine and N-acetyl-D-glucosamine, which feed rates were utilized for different PGC hydrogels to

54 International Journal of Bioprinting (2016)–Volume 2, Issue 1